During mitosis, protein kinases coordinate cellular reorganization and chromosome segregation to ensure accurate distribution of genetic information into daughter cells. Multiple protein kinases contribute to mitotic regulation, modulating molecular signaling more rapidly than possible with gene expression. However, a comprehensive understanding of how kinases regulate mitotic progression remains elusive. The challenge arises from multiple functions and substrates, a large number of "bystander" phosphorylation events, and the brief window in which all mitotic events transpire. Analog-sensitive alleles of protein kinases are powerful chemical genetic tools for rapid and specific interrogation of kinase function. Moreover, combining these tools with advanced proteomics and substrate labeling has identified phosphorylation sites on numerous protein targets. Here, we review the chemical genetic tools available to study kinase function and identify substrates. We describe how chemical genetics can also be used to link kinase function with cognate phosphorylation events to provide mechanistic detail. This can be accomplished by dissecting subsets of kinase functions and chemical genetic complementation. We believe a complete "chemical genetic toolbox" will ultimately allow a comprehensive understanding of how protein kinases regulate mitosis.