Dynamic protein phosphorylation, a major cellular regulatory system, is tightly controlled by coordinating the reversible action of protein kinases and phosphatases. Recent evidence is consistent with sophisticated mechanisms that regulate both kinases and phosphatases in the cell. Dual specificity phosphatases, which act on phosphorylated serine, threonine, and tyrosine residues in proteins, are valid targets for drug discovery. Chemical complementation combines genetic manipulations with chemical biology and high-content multiparametric analyses and was developed as a screening approach to discover protein phosphatase inhibitors. Using a dual specificity mitogen-activated protein kinase phosphatase as an example, a detailed protocol, discussion of issues relating to data analysis, and high-throughput implementation of the chemical complementation approach to drug discovery is presented.