diff --git a/dist/tools/buildsystem_sanity_check/check.sh b/dist/tools/buildsystem_sanity_check/check.sh
index ba9b88d97b..7e5b4bedff 100755
--- a/dist/tools/buildsystem_sanity_check/check.sh
+++ b/dist/tools/buildsystem_sanity_check/check.sh
@@ -188,6 +188,19 @@ check_board_insufficient_memory_not_in_makefile() {
         | error_with_message 'Move BOARD_INSUFFICIENT_MEMORY to Makefile.ci'
 }
 
+# Test applications must not define the APPLICATION variable
+checks_tests_application_not_defined_in_makefile() {
+    local patterns=()
+    local pathspec=()
+
+    patterns+=(-e '^[[:space:]]*APPLICATION[[:space:]:+]=')
+
+    pathspec+=('tests/**/Makefile')
+
+    git -C "${RIOTBASE}" grep "${patterns[@]}" -- "${pathspec[@]}" \
+        | error_with_message "Don't define APPLICATION in test Makefile"
+}
+
 error_on_input() {
     ! grep ''
 }
@@ -200,6 +213,7 @@ all_checks() {
     check_cpu_cpu_model_defined_in_makefile_features
     check_not_setting_board_equal
     check_board_insufficient_memory_not_in_makefile
+    checks_tests_application_not_defined_in_makefile
 }
 
 main() {