The ubiquitin specific protease (USP) enzyme USP9X is amongst the best studied human deubiquitinases (DUBs), with a myriad of described targets and cellular roles. USP9X has been touted as both an oncogene and a tumour suppressor in different cancer contexts, which has confounded the field and questioned its viability as a therapeutic target. Here we describe WEHI-092, a novel piperazine-based USP9X-specific small molecule inhibitor, and map its binding site to the USP9X fingers subdomain, a binding region distinct from known DUB inhibitor binding sites. Using in-depth proteomics and ubiquitinomics, we show that USP9X has a defined set of substrates when compared to USP7, indicating remarkable DUB target specificity. The substrate profile of USP9X varies significantly across cancer cell lines, yet notably, we reveal a core set of 17 proteins commonly regulated by USP9X in most or all cell lines, which we consider as proximal biomarkers for USP9X inhibition. Consistent with our proteomic analyses, we show that WEHI-092 treatment arrests cells in metaphase without inducing cell death, which may account for growth suppression seen in long-term clonogenic assays in most cancer cell lines, and positions USP9X inhibitors as a new potential class of selective mitosis blocking agents.