A novel approach using sheath flow chip-based CE coupled to a quadrupole TOF mass spectrometer was developed and applied to the analysis of an O-glycopeptide. The method provided good separation and sensitive analysis of the glycopeptide and its P-elimination product. The P-elimination reaction of the glycopeptide with ammonia or dimethylamine was adapted for changing the glycopeptide into deglycosylated form and for specifying the site of glycosylation. MS/MS analysis of the native peptide, glycopeptide and P-elimination product enabled the identification of the glycosylation site through the comparison of the fragmentations and peptide sequence analysis.