We describe microchip-based phosphate-affinity electrophoresis (mu PAE) for separation of peptides aimed at determination of kinase activity. The mu PAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form. We synthesized a Phos-tag-poly(dimethylacrylamide) conjugate. The conjugate and the sample solutions were autonomously injected into a PDMS-glass hybrid microchip. The two solutions were contacted together in the microchannel. When the peptides were electrophoresed into the Phos-tag-poly(dimethylacrylamide) region, the phosphorylated peptide was specifically trapped, and separated from the nonphosphorylated peptide in 10 s. The results were quantified by the areas of the fluorescence peaks. The calibration plot obtained with standard samples showed an excellent linearity and a LOD of 0.9% phosphorylated peptide among the total peptides. For c-Src-reacted samples, the results from the mu PAE were in good agreement with those from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The mu PAE was also successful in the presence of inhibitors for c-Src. The measured 50% inhibitory concentration values for staurosporine, PP2, and SU6656 were in good agreement with the literature values.