Protein phosphorylation is the most important reversible post-translational modification in cells. Analysis of phosphorylated proteins and identification of their phosphorylation sites is helpful for understanding their biological functions. MALDI-TOF-MS and ESI-Q-TOF-MS play important roles in protein phosphorylation analysis. In this work, immobilized metal affinity chromatography (IMAC) was used to selectively enrich phosphopeptides from protein digest mixtures, and treatment of phosphopeptides with alkaline phosphatase was used to confirm the phosphorylation. Finally, the phosphorylation sites were determined by tandem mass spectrometry analysis and database searching.