Updated PubMed. Kinase inhibitors often exhibit on/off-target effects, and efficient data analysis is essential for assessing these effects on the proteome. We developed a workflow for rapidly performing such a proteomic assessment, termed as kinase inhibitor proteome impact analysis (KOPI). We demonstrate KOPI’s utility with staurosporine on the leukemic K562 cell proteome. We analysed the changes in the proteome, thermal stability and phosphoproteome in the cells response to staurosporine. We identified systemically staurosporine’s non-kinome interactors, and showed for the first time that it caused paradoxical hyper- and biphasic phosphorylation.