Abstract Because of its versatility (all types of substrates), robustness (Z’>0.8), rapid performance, and its ease of use, the luminescence based Kinase GloTM assays platform have gained wide acceptance in many drug screening programs for protein kinase inhibitors. We have attempted to extend our strategy by now monitoring ADP product formation in the kinase reaction. The new assay we developed (ADP Glo) maintains all the positive features of Kinase GloTM such as universality, and applicability to all kinds of kinase substrates regardless of their nature with no prior modification (peptides, protein, polymer, lipids, and sugars) but In stead of measuring ATP depletion, it monitors ADP production. It detects ADP at early stages of enzyme reactions with very high signal to background (SB) ratio. It can be run at a wide range of ATP concentrations (low micromolar to millimolar), is sensitive to low ADP concentrations (0.1 pmol), and it can be carried out in high density plate formats. This assay is robust and homogenous, and does not require antibodies or custom synthesized substrates. It is ideal to search for optimal substrates in a collection of peptides, proteins, or lipids in a single assay format, and to screen for ATP competitive and Non competitive inhibitors. Because of the high dynamic range and more importantly, the high SB values even at low % ATP to ADP conversion, the assay allows the use of low amounts of enzyme during high-throughput screening. Evaluation of this assay in a screen of a large chemical library showed minimal false hits attesting to the quality of this system. The ADP-GloTM assay is applicable to all types of kinases, ATPases, multidrug resistant proteins such as MDR1-Pgp, helicases, topoisomerases, and heat shock proteins and as well as many other ADP producing enzyme reactions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1048.