Glycosylation profiling is an effective methodology for achieving a comprehensive understanding of glycoproteins and their alterations in a multitude of pathological conditions. However, in comparison to N-glycosylation, O-glycosylation presents significant challenges in terms of both qualitative and quantitative mass spectrometric analysis. A recently developed oxidative release protocol enables the selective formation of O-glycans containing a carboxyl group derived from the amino acid residue. In this study, 3-nitrophenylhydrazine was used to derivatized the common carboxyl group in a mild hydrophilic solution.