Recently, we developed a novel type of phosphate-affinity gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear manganese(II) complex of a phosphate-binding tag nanomolecule, Phos-tag, which enables the mobility shift detection of phosphorylated proteins from their nonphosphorylated counterparts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the quantitative analysis of protein kinase and phosphatase reactions on a polyacrylamide gel without any special apparatuses, radioactive isotopes, or chemical labels. This review article summarizes four applications of protein phosphorylation profiling using a type of affinity electrophoresis, Mn2+ – Phos-tag SDS-PAGE, as follows: i) in vitro kinase activity profiling for the analysis of the phosphoprotein isotypes derived from various kinase reactions, ii) in vivo kinase activity profiling for the analysis of extracellular signal-dependent protein phosphorylation, iii) in vitro kinase inhibition profiling for the quantitative analysis of a kinase-specific inhibitor, and iv) a two-dimensional mobility-shifting procedure using Mn2+ – Phos-tag SDS-PAGE for the detailed analysis of phosphoprotein isotypes. In addition, we describe the significant advantages, including a higher resolution power for the separation of protein phosphoisotypes compared with the conventional gel-based electrophoresis methods. Protein phosphorylation profiling can provide the basis for understanding the molecular origins of diseases and potentially developing tools toward therapeutic intervention. Therefore, the phosphate-affinity gel electrophoresis methodologies established by using Phos-tag can greatly facilitate the phosphoproteomics for the determination of protein phosphorylation status in life science laboratories worldwide.