Glycogen synthase kinase 3beta (GSK3beta) is a well-known marker and potential therapeutic target in non-insulin-dependent diabetes mellitus and Alzheimer's disease. Our recent demonstration that GSK3beta has a previously unrecognized role in colorectal cancer facilitates the development of a nonradioisotopic in vitro kinase assay (NRIKA) for detecting GSK3beta activity in gastrointestinal cancer cells. The NRIKA uses a sequential combination of immunoprecipitations to isolate GSK3beta in sample cells' lysates, and an in vitro kinase reaction that uses recombinant beta-catenin protein (substrate) and nonradioisotopic ATP, followed by immunoblotting to detect beta-catenin phosphorylated in serine 33, 37 and/or threonine 41 residues. The NRIKA detected higher expression of active GSK3beta in stomach, colon, pancreas and liver cancer cell lines than in human embryonic kidney cells (HEK293) considered nonneoplastic. Inhibition of cancer cell-derived GSK3beta activity by GSK3beta inhibitors (SB-216763, AR-A014418) was detected by the NRIKA. GSK3beta inhibition attenuated survival and proliferation and induced apoptosis in all types of cancer cells but not in HEK293. These findings supported the idea that the pathologic roles of GSK3beta are definite and common in various types of cancer. The NRIKA provides a basis for evolving a high-throughput tool for testing substances for GSK3beta inhibition, and for screening and identifying novel GSK3beta inhibitors with a view to discovering drugs for treatment of cancer as well as non-insulin-dependent diabetes mellitus and Alzheimer's disease.