We have developed a chemical proteomic strategy for the systematic interrogation of endogenous phosphoprotein phosphatases (PPP) and their interacting proteins, including regulatory and scaffolding subunits, and substrates (the 'PPPome'). PPPs are captured using an immobilized non-selective PPP inhibitor, followed by identification and quantification by mass spectrometry. Using this approach, we map the PPPome in human cancer cell lines, mouse tissues, and yeast species, identify cell and tissue type specific PPP expression patterns, and discover new PPP interacting proteins.