Ubiquitylation is a protein modification implicated in several cellular processes. This process is reversible by the action of deubiquinating enzymes (DUBs). USP45 is a ubiquitin specific protease about which little is known, aside from roles in DNA damage repair and differentiation of the vertebrate retina. Here, by using mass spectrometry we have identified Spindly as a new target of USP45. Our data show that Spindly and USP45 are part of the same complex and that their interaction specifically depends on the catalytic activity of USP45. In addition, we describe the type of ubiquitin chains associated with the complex that can be cleaved by USP45, with a preferential activity on K48 ubiquitin chain type and potentially K6. Here, we also show that Spindly is mono-ubiquitylated and this can be specifically removed by USP45 in its active form but not by the catalytic inactive form. Lastly, we identified a new role for USP45 in cell migration, similar to that which was recently described for Spindly.