Updated publication reference for PubMed record(s): 30459171, 32681153. Protein glycosylation is one of the most common protein modifications and plays essential roles in biology and therapeutics. However, the analysis of in vivo O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of technology. Here, a chemoenzymatic method was presented for the site-specific extraction of O-linked glycopeptides (EXoO), which achieved simultaneous enrichment and unambiguous mapping of over 3,000 O-linked glycosylation sites and corresponding O-linked glycans on over 1,000 proteins in human kidney tissues, serum and T cells. The large-scale localization of O-linked glycosylation sites nearly doubles the sites identified in the last decades demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Structural analysis of the sites revealed conserved motifs and topological orientation facing extracellular space or lumen of ER and Golgi. Striking signature of aberrant in vivo O-linked glycoproteome was observed between kidney tumor and normal tissues discovering key factors in tumor biology. The O-linked glycoproteome play diverse roles on the ER, Golgi membrane, cell surface and extracellular space arguing that EXoO can be applied broadly to the analysis of O-linked glycoproteins in biology and therapeutics.