O‐Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post‐translational modifications. Compared with N‐linked glycosylation, O‐glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O‐glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O‐glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O‐glycosylation, i.e. O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O‐glycosylation. For the large‐scale analysis of mucin‐type glycosylation, the glycan simplification strategies including the ‘SimpleCell’ technology was introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O‐N‐acetylgalactosamine and O‐N‐acetylglucosamine glycosylation.
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