Reversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting cellular phosphorylation activity with okadaic acid, microcystin-LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.