Detailed characterization of O-glycoproteins is imperative to answering questions related to how glycosylation varies as a function of disease state. As demonstrated for analysis of glycopeptides produced by SmE digestion of O-glycoproteins TIM-1, MUC-16 and MUC-1, UVPD excels in providing structural insight about the linkages of individual O-glycans and elucidates overall patterns of glycosylation, even revealing in some cases the number of occurrences of certain glycan linkages. Harvesting the full potential of UVPD mass spectra will require development of more advanced software tools to help streamline the data interpretation workflow, as manual validation of the highly heterogeneous O-glycoproteoforms of mucin proteins is incredibly tedious. Nonetheless, the combination of SmE mucinase and UVPD has proved effective for O-glycopeptide characterization, allowing confirmation of a variety of structures for large and small glycoforms alike. As new software tools emerge, this integrated workflow should be applicable to analysis of complex O-glycoproteomes in a high-throughput manner.