Updated project metadata. The hemostatic system comprises platelet aggregation, coagulation and fibrinolysis, and is critical to the maintenance of vascular integrity. Multiple studies indicate that glycans play an important role in maintaining the hemostatic system, however, most investigations have focused on N-glycans and little is known about the location and function of O-glycans. Here we performed the first systematic analysis of hemostatic O-glycosylation using lectin affinity chromatography coupled to LC-MS/MS to determine the precise location of O-glycans in human plasma, platelets, and endothelial cells. We identified the hitherto largest O-glycoproteome from native tissue, demonstrating that O-glycosylation is a ubiquitous modification of hemostatic proteins.