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The functional impact of glycosylation on drug efficacy and safety profiles has been demonstrated in a wide range of biopharmaceuticals. Understanding of the N-glycosylation pathway and the advent of analytical technologies has enabled both detailed and rapid characterization of N-glycans, thus providing critical insights for regulatory compliance. In comparison, O-glycosylation encompasses several types of protein modifications by a more heterogeneous pool of sugars, and the understanding of its biology is less mature. Concomitantly, there exist several limitations in O-glycan analytical strategies, impacting both O-glycoprotein-based drug development and regulatory compliance. Here, we aim to provide a critical review of the biology and functional importance of O-glycosylation in the context of existing and potential O-glycoprotein drugs, as well as the corresponding analytical methods. The ultimate goal is to identify the gaps in current analytical methods and propose potential future directions for O-glycosylation analysis, to support the development and production of O-glycosylated biopharmaceuticals.