Updated project metadata. Successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes. Biochemical analysis of cytokinesis is challenging due to its dynamic and rapid nature that necessitates the establishment of robust methodologies. Our comparative proteomic approach suggests that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-suited system for global biochemical analysis in mammalian cells. We established a phosphoproteomic signature of cytokinesis. More than 10,000 phosphorylation sites were systematically screened and monitored; around 800 of these phosphosites were up-regulated during cytokinesis.