Protein phosphorylation is one of post-translational modifications (PTM), which plays a role in regulation of development, signal transduction, and processes of diseases. Because of high sensitivity, considerable throughput, and residue resolution, mass spectrometry (MS) has been the most popular tool for phosphorylation modification analysis. Common MS-centric phosphoproteomics workflow includes phosphorylation modified peptides sampling, LC-MS/MS detection, phosphorylation sites assignment and quantification. We summarized and discussed these workflow parts in this review.