The mammalian microtubule-associated serine/threonine (MAST) kinases are a highly conserved subfamily of AGC kinases that are implicated as therapeutic targets for cancer and diabetes. However, the activity, regulation, and substrates of MAST kinases are poorly understood. Stable 18O-ATP labeled kinase assay linked phospho-proteomics (SIKALIP) identifies a collection of putative Mast2 substrate. Our results develop a biochemical profile of the MAST Kinases, provide insight into their regulatory mechanisms, and begin to identify the cellular functionof MAST2.