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Protein serine/threonine phosphatase (PP2A) is a major cellular enzyme implicated in the control of numerous signaling processes. The accurate measurement of PP2A activity in crude cell lysates, immune complexes, and purified preparations provides insight into the function and regulation of this essential enzyme, which, in turn, can lead to a better understanding of the signaling pathways that it modulates. The method presented here utilizes 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) and a FLEXstation for the continuous measure of PP2A activity associated with many different protein preparations. This automated fluorescence-based assay offers several distinct advantages over colorimetric and radioactive assays of phosphatase activity including (1) decreased substrate preparation time, (2) real-time kinetic data, (3) high sensitivity, and (4) the capability to analyze a wide variety of phosphatases.