Post-translational modifications composed of ubiquitin regulate a wide range of cellular functions and are tightly controlled by deubiquitinating enzymes (DUBs). The recent start of clinical trials with inhibitors of three DUBs highlights the therapeutic potential of their modulation in cancer and neurodegenerative diseases. However, how DUBs decode ubiquitin signals and how they are engaged by small molecule ligands remain poorly understood at the molecular level. My group uses an integrated chemical and structural biology approach to shed light on how DUBs function and how they can be specifically inhibited. In my talk, I will discuss discuss how protein-based as well as small molecule-based chemical probes can be used to interrogate DUB function and activity regulation: I will firstly focus on how protein-based probes have enabled the discovery of novel activities in DUBs. Secondly, I will introduce covalent-reversibly acting small molecule USP30 activity sensors suitable for live cell applications. Using these reagents, I will show how activity of the mitophagy-associated DUB USP30 is regulated by in a cell state-dependent manner. These approaches complement our recently published chimeric protein engineering approach which revealed how USP30 can be specifically inhibited and how this provided a framework for specific DUB inhibition. Collectively, I will highlight how ubiquitin signalling in cells can be deciphered with chemical biology using protein-based and small molecule chemical probes.