Abstract The current interest in protein homeostasis as a therapeutic strategy has highlighted the importance of protein ubiquitination as a post-translational modification. Protein degradation has been enabled by several approaches (e.g. small molecule degraders, molecular glues, and PROTACsTM). Deubiquitinating enzymes (DUBs) are proteases that catalyse the de-ubiquitination of protein substrates and as such offer an alternative way to regulate protein homeostasis. Furthermore, since ubiquitination also controls a plethora of regulatory functions beyond direct degradation, inhibition of DUBs provides additional opportunities to manipulate critical cellular processes. As a result of this dual role in protein degradation and signalling, as well as their increasing linkage to the etiology of numerous pathological conditions including cancer and neurodegeneration, DUBs have emerged as an attractive target class for the development of first-in-class medicines with high therapeutic impact. However until recently, DUBs have proven largely refractory to drug discovery efforts despite significant industry efforts. We have developed a purpose-built drug discovery platform (UbiPlex™) for the identification and development of DUB inhibitors. Herein, we will highlight significant recent developments to this platform including the addition of multiple DUB crystal structures (including fragments and small molecule co-crystals), biophysics, chemo/bio-informatics capabilities and focussed screening libraries. These developments have allowed the identification of novel, highly potent (e.g. IC50 < 100 nM), reversible inhibitors with drug like properties against multiple therapeutically relevant DUB targets. Selectivity profiles of representative inhibitors against panels of DUBs and other non-related enzymes (e.g. kinases, proteases) will be described, demonstrating that excellent selectivity is achievable. Finally, we will describe our progress towards the development of lead molecules with drug-like properties with the aim of rapidly establishing proof-of-concept studies in vivo for previously unexplored DUB targets. This work exemplifies the broad tractability and druggability of the DUBome and provides an alternative strategy for manipulation of the UPS. Furthermore, these potent, selective, cell penetrant molecules represent important chemical probes for pharmacological validation of the biological pathways associated with deubiquitinase inhibition as well as starting points for the development of new therapeutics for cancer and associated disorders. Citation Format: Tim Harrison, Xavier Jacq, Colin O'Dowd, Gerald Gavory, Oliver Barker, Christina Bell, Frank Burkamp, Stephanie Burton, Eamon Cassidy, Matthew Helm, Peter Hewitt, Natalie Page, Shane Rountree, Ewelina Rozycka, Steven Shepherd, Steven Whitehead, Mark Wappett. Targeting the ubiquitin-proteasome system by small molecule inhibition of the DUBome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-049.