Significance Ubiquitination involves the covalent attachment of the protein ubiquitin to substrates. It can be reversed by the action of deubiquitinating enzymes (DUBs), thereby providing an important layer of regulation. Originally believed to be restricted to lysine residues, it is emerging that additional amino acids, including serine, threonine and cysteine, are also modified. It remains unknown which DUBs might target these unusual sites for deubiquitination. Herein, we develop representative model substrates and screen 53 DUBs for non-lysine activity, thereby providing important insights into DUB function. Strikingly, we find that a poorly studied DUB class has potent and highly selective serine/threonine activity. These findings suggest that non-lysine ubiquitination rivals the regulatory sophistication of its conventional counterpart and might serve distinct cellular functions.