diff --git a/lib/galaxy/dependencies/dev-requirements.txt b/lib/galaxy/dependencies/dev-requirements.txt
index a59d5b4e25f..28c26a49138 100644
--- a/lib/galaxy/dependencies/dev-requirements.txt
+++ b/lib/galaxy/dependencies/dev-requirements.txt
@@ -210,7 +210,6 @@ total-perspective-vortex==3.1.3
 trio==0.33.0
 trio-websocket==0.12.2
 tuspy==1.1.0
-twill==3.3.1
 twine==6.2.0
 types-cachetools==6.2.0.20251022
 types-requests==2.32.4.20260107
diff --git a/lib/galaxy/dependencies/pinned-test-requirements.txt b/lib/galaxy/dependencies/pinned-test-requirements.txt
index e9d19429d0e..79ce33341c0 100644
--- a/lib/galaxy/dependencies/pinned-test-requirements.txt
+++ b/lib/galaxy/dependencies/pinned-test-requirements.txt
@@ -162,7 +162,6 @@ total-perspective-vortex==3.1.3
 trio==0.33.0
 trio-websocket==0.12.2
 tuspy==1.1.0
-twill==3.3.1
 types-cachetools==6.2.0.20251022
 types-requests==2.32.4.20260107
 typing-extensions==4.15.0
diff --git a/pyproject.toml b/pyproject.toml
index 4d14622589e..816ceaff448 100644
--- a/pyproject.toml
+++ b/pyproject.toml
@@ -152,7 +152,6 @@ test = [
     "testfixtures",
     "total-perspective-vortex>=3.1.1,<4",  # https://github.com/galaxyproject/total-perspective-vortex/pull/173
     "tuspy",
-    "twill>=3.2.5",  # Python 3.13 support
     "watchdog",
     "Werkzeug",
 ]