Abstract Regulated protein turnover is primarily controlled by the ubiquitin-proteasome system. The only marketed drug related to the ubiquitin-proteasome system, bortezomib, is acting as a proteasome inhibitor and has been approved for the treatment of some hematological cancers. Targeting the upstream ubiquitin conjugation/deconjugation system carries out promises of therapeutics with increased specificity and selectivity. Ubiquitin-specific proteases (USP) are involved in the deubiquitination of specific target substrates regulating their stability, subcellular localization and/or activation status. USP represent a drugable target class due to their thiol-protease catalytic core which is amenable to pharmacological inhibition by small molecules. A genome-wide RNAi screen of the catalytically active human USPs in cancer-relevant cellular models and phenotypic assays allowed us to identify USP7/HAUSP as promising cancer target. Fluorescence-based screening assays using optimized USP substrates including various ubiquitin derivatives (ubiquitin precursor, branched ubiquitin chains) as well as specific, physiological substrates were developed. High-throughput screening performed on our chemically diverse library followed by different optimization programs resulted in the discovery of several series as novel USP7 inhibitors. Our progress made towards the specificity issue will be presented here with the identification of the first USP7-specific series. These will help further validate this novel class of molecular targets and may provide a structural basis for the development of new anticancer drugs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 2642.