Herein, we demonstrate the separation of phosphoprotein isotypes having the same number of phosphate groups using phosphate-affinity SDS-PAGE. The phosphate-affinity site is a polyacrylamide-bound Phos-tag that enables the mobility shift detection of phosphoproteins from their nonphosphorylated counterparts. As the first practical example of the separation, we characterized the monophosphorylated Tau isotypes by each of three tyrosine kinases, c-Abl, MET, and Fyn. Each monophosphoisotype phosphorylated at the Tyr-394, Tyr-197, or Tyr-18 was detected as three distinct migration bands. As a further application, we extended this technique to the mobility shift analysis of His and Asp phosphoisotypes in the Sinorhizobium meliloti FixL/FixJ two-component system. FixL is autophosphorylated at the His-285 with ATP, and the phosphate group is transferred to the Asp-54 of FixJ and subsequently removed by the FixL phosphatase activity. Using this method, we first performed simultaneous detection of the phosphorylated and nonphosphorylated isotypes of FixL and FixJ generated in their phosphotransfer reaction in vitro. As a result, a monophosphoisotype of FixL containing the phosphorylated His residue was confirmed. As for FixJ, on the other hand, two monophosphoisotypes were detected as two distinct migration bands. One is a well-known isotype phosphorylated at the Asp-54. The other is a novel isotype phosphorylated at the His-84.