This study presents a comprehensive quantitative atlas of mitotic phosphorylation in human cells. Using stable isotope labeling and advanced phosphopeptide enrichment techniques, the authors identified over 14,000 unique phosphorylation events, more than half of which have not been previously described. The analysis revealed that over 1,000 proteins exhibit increased phosphorylation during mitosis, many of which are known cell cycle regulators. The majority of these regulated phosphorylation sites are located in [S/T]P motifs, suggesting that they are direct targets of cyclin-dependent kinases (CDKs). The study also identified two novel motifs that suggest the presence of undiscovered mitotic kinases. The findings provide significant insights into the regulatory mechanisms of the cell cycle and highlight the importance of phosphorylation in mitotic processes.This study presents a comprehensive quantitative atlas of mitotic phosphorylation in human cells. Using stable isotope labeling and advanced phosphopeptide enrichment techniques, the authors identified over 14,000 unique phosphorylation events, more than half of which have not been previously described. The analysis revealed that over 1,000 proteins exhibit increased phosphorylation during mitosis, many of which are known cell cycle regulators. The majority of these regulated phosphorylation sites are located in [S/T]P motifs, suggesting that they are direct targets of cyclin-dependent kinases (CDKs). The study also identified two novel motifs that suggest the presence of undiscovered mitotic kinases. The findings provide significant insights into the regulatory mechanisms of the cell cycle and highlight the importance of phosphorylation in mitotic processes.