Ubiquitination is a key posttranslational modification, which affects numerous biological processes and is reversed by a class of enzymes known as deubiquitinases (DUBs). This family of enzymes cleaves mono-ubiquitin or poly-ubiquitin chains from a target protein through different mechanisms and mode of interactions with their substrates. Studying the role of DUBs in health and diseases has been a major goal for many laboratories both in academia and in industry. However, the field has been challenged by the difficulties in obtaining native substrates and novel reagents using traditional enzymatic and molecular biology approaches. Recent advancements in the synthesis and semisynthesis of proteins made it possible to prepare several unique ubiquitin conjugates to study various aspects of DUBs such as their specificities and structures. Moreover, these approaches enable the preparation of novel activity based probes and assays to monitor DUB activities in vitro and in cellular contexts. Efforts made to bring new chemical entities for the selective inhibition of DUBs based on these tools are also highlighted with selected examples.