Osteoarthritis (OA) is the most common rheumatic pathology. Because currently available diagnostic methods are limited and lack sensitivity, the identification of new specific biological markers for OA has become a focus. The purpose of this study was to identify novel protein biomarkers for moderate and severe OA in serum. Sera were obtained from 50 moderate OA patients, 50 severe OA patients, and 50 nonsymptomatic controls. Serum protein levels were analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) and matrix-assisted laser desorption/ionization (MALDI)-TOF/TOF mass spectrometry. We identified 349 different proteins in the sera, 262 of which could be quantified by calculation of their iTRAQ ratios. Three sets of proteins were significantly (p < 0.05) changed in OA samples compared to controls. Of these, 6 were modulated only in moderate OA, 13 only in severe OA and 7 in both degrees. Although some of these proteins, such as cartilage oligomeric matrix protein, have a previously reported putative biomarker value for OA, most are novel biomarker candidates for the disease. These include some complement components, lipoproteins, von Willebrand factor, tetranectin, and lumican. The specificity and selectivity of these candidates need to be validated before new molecular diagnostic or prognostic tests for OA can be developed.