Updated publication reference for PubMed record(s): 30713027. The multi-domain deubiquitinase USP15 regulates diverse eukaryotic processes and has been implicated in numerous diseases. We developed ubiquitin variants (UbVs) that targeted either the catalytic domain or each of three adaptor domains in USP15, including the N-terminal DUSP domain. Taking advantage of the modular nature of ubiquitin, we designed a linear dimer (diUbV) consisting of a UbV that bound the DUSP domain followed by a UbV that bound the catalytic domain, which exhibited enhanced specificity and more potent inhibition of USP15 catalytic activity than either UbV alone. In cells, the UbVs inhibited the deubiquitination of two USP15 substrates, SMURF2 and TRIM25, and the diUbV inhibited the effects of USP15 on the TGF- pathway. Structural analyses of three distinct UbVs bound to the catalytic domain revealed that the inhibitors locked the active site in a closed, inactive conformation. The structure of a UbV-DUSP domain complex revealed that the UbV formed an unusual strand-swapped dimer that bound two DUSP domains. These inhibitors will enable the study of USP15 function in vitro and in vivo to explore the role of the enzyme in oncology, neurology, immunology and inflammation.