Lubricin or proteoglycan-4 (PRG-4) is a mucinous glycoprotein that lubricates the cartilage and maintains normal tissue function and cell homeostasis. Altered O- glycoproteforms of lubricin have been found in osteoarthritis (OA) synovial fluid (SF), which could ostensibly be used as a biomarker to diagnose early onset OA. However, SF is invasive to obtain and generally would not be surveyed on otherwise healthy individuals. Thus, a plasma-based OA screening tool focused on lubricin glycosylation could serve as an improved biomarker. In this report, we used glycomics and glycoproteomics to identify glycoproteoforms of OA lubricin in SF and plasma. We obtained near-complete sequence coverage of the mucin domain of lubricin and its glycosylation using matched SF/plasma from OA patients (N=5). In SF we observed a spectrum of O-glycans on lubricin ranging from the single GalNAc monosaccharide up to branched pentasaccharide and low in sialylation compared lubricin in plasma, decorated with sialylated Gal1-3GalNAc. We also detected splice variant-specific peptides from lubricin non-glycosylated region showing that the longest splice form of lubricin was exclusively present in SF, while in addition shorter splice variants were also present in plasma.Following elution, the mucin-enriched plasma or synovial fluid was subjected to reduction, alkylation, and proteolytic digestion. To begin, dithiothreitol (DTT, Sigma) was added to a concentration of 2 mM and reacted at 65 °C for 20 min followed by alkylation in 5 mM iodoacetamide (IAA, sigma) for 15 min in the dark at RT. Subsequently, mucinase SmE was added at an enzyme:substrate (E:S) ratio of 1:20 and allowed to react overnight at 37 °C. At this point, the samples were split into two aliquots, one containing 90% and the other containing the remaining 10%. The former represented the “mucinase-only” digest that did not undergo any further proteolysis. The latter was subjected to an additional digestion with trypsin at a 1:50 E:S ratio for 6 hours at 37 °C. All reactions were quenched by adding 1 µL of formic acid (Thermo Scientific) and diluted to a volume of 200 µL prior to desalting. Addition of formic acid also caused sodium deoxycholate to precipitate out of solution, and the resulting supernatant was transferred to a new tube before desalting. Desalting was performed using 10 mg Strata-X 33 µm polymeric reversed phase SPE columns (Phenomenex). Each column was activated using 500 µL acetonitrile (ACN) (Honeywell) followed by of 500 µL of 0.1% formic acid, 500 µL of 0.1% formic acid in 40% ACN, and equilibration with two additions of 500 µL of 0.1% formic acid. After equilibration, the samples were added to the column and rinsed twice with 200 µL of 0.1% formic acid. The columns were transferred to a 1.5 mL tube for elution by two additions of 150 µL of 0.1% formic acid in 40% ACN. The eluent was then dried using a vacuum concentrator (LabConco) prior to reconstitution in 10 µL of 0.1% formic acid.