Protein phosphorylation is a universal regulatory way in organism, and it is a key event in cell signal transduction. Mass spectrometry has emerged as an increasingly viable tool for this task. The methodologies currently available for the analysis of phosphoproteins by mass spectrometry were summarized, including enrichment of compounds of interest using immobilized metal affinity chromatography, antibody and chemical tagging techniques, detection of phosphopeptides using mass mapping and precursor ion sequencing, localization of phosphorylation sites by peptide sequencing, and quantitation of phosphorylation by the introduction of mass tags. Despite the variety of powerful analytical methods that are now available, complete characterization of the phosphorylation state of a protein isolated in small quantities from a biological sample remains far from routine.