Phosphorylation is one of the most important post-translational protein modifications, which is related to the regulation of many activities of life． As a sort of reaction catalyzed by protein kinases, it functions significantly in cell signaling. To keep up with the situation that protein phosphorylation analysis and phosphorylated site identification have been a focus of the current proteomic study, and benefit the functional dissection on various protein phosphorylation reactions, this article introduces the progress in the studies of main types and functions of protein phosphorylation, the identification of the phosphopeptides, the determination and prediction of specific phosphorylation sites. As a focal point, protocols based on isotope labeling, chemilum inescence, etc, are elucidated. Western blotting has been proven sensitive and specific in the identification of phosphoproteins, based on which, there have appeared some improved approaches, such as NanoPro100/1000 ultramicro-protein analysis system. Phosphorylation analytic tools and softwares are being swiftly developing. According to numerous researches, protein phosphorylation/dephosphorylation modifications are important with many aspects of meaning. Controlling the cellular endogenous enzymic 'activities', protein phosphorylation/dephosphorylation are more able to rapidly react to external stimulus than enzymic re-synthesis/decomposition. A series of phosphorylation/dephosphorylation reactions in different tissues and with different temporal and spatial characteristics ensure the sustained responses of cells to external signals, and play a role in the cascade amplifying for the external signals. Protein phosphorylation/dephosphorylation study will continue to be a hotspot in the exporation of life activity regulatory mechanism.