A number of assays have been developed for high-throughput screening (HTS) of potentially bioactive compounds. To screen millions of chemical compounds efficiently, the best detection technology prior to initiating HTS must be chosen. Ideally, a non-radioisotope (non-RI), homogeneous method, equivalent to the most reliable assay for a particular target, should be selected as an HTS method. Protein kinases are among the most important classes for drug discovery because they participate in various signaling pathways. Several HTS technologies are available for kinase activity: SPA (Amersham, Piscataway, NJ, U.S.A.), HTRF (CIS-US, Inc., Bedford, MA, U.S.A.), IMAP (Molecular Devices, Sunnyvale, CA, U.S.A.), and Z'-LYTE (Invitrogen, Carlsbad, CA, U.S.A.). The amount of phosphorylated product is detected by different methods in these assays. Recently, Kinase-Glo Luminescent Kinase Assay, a non-RI, homogeneous, adenosine triphosphate (ATP) quantitative kit useful for kinase activity detection, has become available from Promega (Madison, WI, U.S.A.). ATP is a universal substrate for kinases. Thus, the Kinase-Glo assay shows promise for becoming the primary method of determining kinase activity in HTS. We have developed a Kinase-Glo system for cyclin-dependent kinase 4 (Cdk4), and compare its results with those of the filtration method, the most reliable assay for in vitro Cdk4 activity. In addition, the reliability and sensitivity of the Kinase-Glo are discussed.