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Protein phosphorylation plays a key role in the regulation of most cellular processes and the disorder of this process often leads to illness or even cancers. Panoramic phosphorylation analysis of organisms known as “phosphoproteomics” has become more important and popular in scientific research fields these days due to its ability of gaining significant information about phosphorylation events and its assistance in understanding how those regulatory functions work in cell. Analytical strategies have been developed for comprehensive analysis of phosphoproteome over the past several years on the aspects of both profiling and quantification. In this review, we will discuss some of the recent applications of phosphoproteomics by using several commonly used technologies, and the applications on study of cell signal transduction pathways and disease-related biomarker and drug targets discovery will also be covered.