Deubiquitinating enzymes (DUBs) control the removal of ubiquitin and ubiquitin-like proteins from cellular proteins. There are approximately 100 DUBs in the human genome, and they regulate diverse biochemical, cellular and physiological processes. Notably, they are known to control many pathways which are misregulated and affected in human diseases, such as cancer, immunology and neurodegeneration. Due to the broad scope of DUB biology, they are emerging as a target class for inhibitor development. In this chapter, we will describe the promise of targeting DUB in different disease contexts, describe practices for identifying and validating small-molecule inhibitors and physiologically relevant substrates of DUBs and review recent examples of well-characterized DUB inhibitors. These advances underscore the excitement and potential in targeting DUBs for both therapeutic and research purposes.