Protein phosphorylation constitutes a major type of post-translational modifications mobilizing a high number of genes. It is involved in many crucial cell processes and largely participates in the features of the proteome. For several biological and technical reasons, the characterization of protein phosphorylation requires a combination of distinct procedures. In this review, a special emphasis is given to analytical strategies connected with the determination of the presence of phosphorylation sites and their localization by mass spectrometry (MS). The feasibility of selected combinations of analytical approaches for diverse objectives of phosphoproteomic research is discussed.