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Reversible protein phosphorylation mediated by kinases, phosphatases, and regulatory molecules is an essential mechanism of signal transduction in living cells. Although phosphorylation is the most intensively studied of the several hundred known posttranslational modifications on proteins, until recently the rate of identification of phosphorylation sites has remained low. The use of tandem mass spectrometry has greatly accelerated the identification of phosphorylation sites, although progress was limited by difficulties in phosphoresidue enrichment techniques. We have improved upon existing immobilized metal-affinity chromatography (IMAC) techniques for capturing phosphopeptides, to selectively purify phosphoproteins from complex mixtures. Combinations of phosphoprotein and phosphopeptide enrichment were more effective than current single phosphopeptide purification approaches. We have also implemented iterative mass spectrometry-based scanning techniques to improve detection of phosphorylated peptides in these enriched samples. Here, we provide detailed instructions for implementing and validating these methods together with analysis by tandem mass spectrometry for the study of phosphorylation at the mammalian synapse. This strategy should be widely applicable to the characterization of protein phosphorylation in diverse tissues, organelles, and in cell culture.