Deubiquitinases (DUBs) are specialized proteases that remove ubiquitin from substrates or cleave within ubiquitin chains to regulate ubiquitylation and therefore play important roles in eukaryotic biology. Dysregulation of DUBs is implicated in several human diseases, highlighting the importance of DUB function. In addition, many pathogenic bacteria and viruses encode and deploy DUBs to manipulate host immune responses and establish infectious diseases in humans and animals. Hence, therapeutic targeting of DUBs is an increasingly explored area that requires an in-depth mechanistic understanding of human and pathogenic DUBs. In this review, we summarize the multiple layers of regulation that control autoinhibition, activation, and substrate specificity of DUBs. We discuss different strategies to inhibit DUBs and the progress in developing selective small-molecule DUB inhibitors. Finally, we propose a classification system of DUB inhibitors based on their mode of action.