diff --git a/crates/red_knot_python_semantic/resources/mdtest/unary/instance.md b/crates/red_knot_python_semantic/resources/mdtest/unary/invert_add_usub.md
similarity index 95%
rename from crates/red_knot_python_semantic/resources/mdtest/unary/instance.md
rename to crates/red_knot_python_semantic/resources/mdtest/unary/invert_add_usub.md
index 85f80c3b8c..98d9c9b1c0 100644
--- a/crates/red_knot_python_semantic/resources/mdtest/unary/instance.md
+++ b/crates/red_knot_python_semantic/resources/mdtest/unary/invert_add_usub.md
@@ -1,4 +1,6 @@
-# Unary Operations
+# Invert, UAdd, USub
+
+## Instance
 
 ```py
 from typing import Literal
diff --git a/crates/red_knot_python_semantic/resources/mdtest/unary/not.md b/crates/red_knot_python_semantic/resources/mdtest/unary/not.md
index f43b486c24..a193437328 100644
--- a/crates/red_knot_python_semantic/resources/mdtest/unary/not.md
+++ b/crates/red_knot_python_semantic/resources/mdtest/unary/not.md
@@ -113,3 +113,101 @@ reveal_type(not ())  # revealed: Literal[True]
 reveal_type(not ("hello",))  # revealed: Literal[False]
 reveal_type(not (1, "hello"))  # revealed: Literal[False]
 ```
+
+## Instance
+
+Not operator is inferred based on
+<https://docs.python.org/3/library/stdtypes.html#truth-value-testing>. An instance is True or False
+if the `__bool__` method says so.
+
+At runtime, the `__len__` method is a fallback for `__bool__`, but we can't make use of that. If we
+have a class that defines `__len__` but not `__bool__`, it is possible that any subclass could add a
+`__bool__` method that would invalidate whatever conclusion we drew from `__len__`. So instances of
+classes without a `__bool__` method, with or without `__len__`, must be inferred as unknown
+truthiness.
+
+```py
+class AlwaysTrue:
+    def __bool__(self) -> Literal[True]:
+        return True
+
+# revealed: Literal[False]
+reveal_type(not AlwaysTrue())
+
+class AlwaysFalse:
+    def __bool__(self) -> Literal[False]:
+        return False
+
+# revealed: Literal[True]
+reveal_type(not AlwaysFalse())
+
+# We don't get into a cycle if someone sets their `__bool__` method to the `bool` builtin:
+class BoolIsBool:
+    __bool__ = bool
+
+# revealed: bool
+reveal_type(not BoolIsBool())
+
+# At runtime, no `__bool__` and no `__len__` means truthy, but we can't rely on that, because
+# a subclass could add a `__bool__` method.
+class NoBoolMethod: ...
+
+# revealed: bool
+reveal_type(not NoBoolMethod())
+
+# And we can't rely on `__len__` for the same reason: a subclass could add `__bool__`.
+class LenZero:
+    def __len__(self) -> Literal[0]:
+        return 0
+
+# revealed: bool
+reveal_type(not LenZero())
+
+class LenNonZero:
+    def __len__(self) -> Literal[1]:
+        return 1
+
+# revealed: bool
+reveal_type(not LenNonZero())
+
+class WithBothLenAndBool1:
+    def __bool__(self) -> Literal[False]:
+        return False
+
+    def __len__(self) -> Literal[2]:
+        return 2
+
+# revealed: Literal[True]
+reveal_type(not WithBothLenAndBool1())
+
+class WithBothLenAndBool2:
+    def __bool__(self) -> Literal[True]:
+        return True
+
+    def __len__(self) -> Literal[0]:
+        return 0
+
+# revealed: Literal[False]
+reveal_type(not WithBothLenAndBool2())
+
+# TODO: raise diagnostic when __bool__ method is not valid: [unsupported-operator] "Method __bool__ for type `MethodBoolInvalid` should return `bool`, returned type `int`"
+# https://docs.python.org/3/reference/datamodel.html#object.__bool__
+class MethodBoolInvalid:
+    def __bool__(self) -> int:
+        return 0
+
+# revealed: bool
+reveal_type(not MethodBoolInvalid())
+
+# Don't trust a possibly-unbound `__bool__` method:
+def get_flag() -> bool:
+    return True
+
+class PossiblyUnboundBool:
+    if get_flag():
+        def __bool__(self) -> Literal[False]:
+            return False
+
+# revealed: bool
+reveal_type(not PossiblyUnboundBool())
+```
diff --git a/crates/red_knot_python_semantic/src/types.rs b/crates/red_knot_python_semantic/src/types.rs
index b61f848daa..2b23469408 100644
--- a/crates/red_knot_python_semantic/src/types.rs
+++ b/crates/red_knot_python_semantic/src/types.rs
@@ -1197,14 +1197,40 @@ impl<'db> Type<'db> {
                 // TODO: see above
                 Truthiness::Ambiguous
             }
-            Type::Instance(InstanceType { class }) => {
-                // TODO: lookup `__bool__` and `__len__` methods on the instance's class
-                // More info in https://docs.python.org/3/library/stdtypes.html#truth-value-testing
-                // For now, we only special-case some builtin classes
+            instance_ty @ Type::Instance(InstanceType { class }) => {
                 if class.is_known(db, KnownClass::NoneType) {
                     Truthiness::AlwaysFalse
                 } else {
-                    Truthiness::Ambiguous
+                    // We only check the `__bool__` method for truth testing, even though at
+                    // runtime there is a fallback to `__len__`, since `__bool__` takes precedence
+                    // and a subclass could add a `__bool__` method. We don't use
+                    // `Type::call_dunder` here because of the need to check for `__bool__ = bool`.
+
+                    // Don't trust a maybe-unbound `__bool__` method.
+                    let Symbol::Type(bool_method, Boundness::Bound) =
+                        instance_ty.to_meta_type(db).member(db, "__bool__")
+                    else {
+                        return Truthiness::Ambiguous;
+                    };
+
+                    // Check if the class has `__bool__ = bool` and avoid infinite recursion, since
+                    // `Type::call` on `bool` will call `Type::bool` on the argument.
+                    if bool_method
+                        .into_class_literal()
+                        .is_some_and(|ClassLiteralType { class }| {
+                            class.is_known(db, KnownClass::Bool)
+                        })
+                    {
+                        return Truthiness::Ambiguous;
+                    }
+
+                    if let Some(Type::BooleanLiteral(bool_val)) =
+                        bool_method.call(db, &[*instance_ty]).return_ty(db)
+                    {
+                        bool_val.into()
+                    } else {
+                        Truthiness::Ambiguous
+                    }
                 }
             }
             Type::KnownInstance(known_instance) => known_instance.bool(),