Investigation of site-specific protein O-glycosylation remains a formidable task in post-translational modification-centred proteomics. In particular, the determination of O-glycosylated amino acids in mucin-like glycopeptides lags far behind the techniques for phosphorylation site and N-glycosylation site identification, for which well-established enrichment techniques are available. The present work investigated beta-elimination of mucin-like O-glycopeptides with a mild alkylamine base and concomitant Michael-type addition using 2-mercaptoethanol as nucleophile applied to synthetic GalNAcylated O-glycopeptides as well as exoglycosidase-treated endogenous peptides isolated from human blood plasma. This strategy permits O-glycosylated sites to be unambiguously localized, even in multiple-glycosylated peptides. Peptides covalently modified with the glycan surrogate exhibit excellent backbone fragmentation in MS/MS due to their stability during CID.