More than a decade after a Nobel Prize was awarded for the discovery of the ubiquitin-proteasome system and clinical approval of proteasome and ubiquitin E3 ligase inhibitors, first-generation deubiquitylating enzyme (DUB) inhibitors are now approaching clinical trials. However, although our knowledge of the physiological and pathophysiological roles of DUBs has evolved tremendously, the clinical development of selective DUB inhibitors has been challenging. In this Review, we discuss these issues and highlight recent advances in our understanding of DUB enzymology and biology as well as technological improvements that have contributed to the current interest in DUBs as therapeutic targets in diseases ranging from oncology to neurodegeneration.