Osteoarthritis (OA) is a common chronic disease, causing joint pain and reduced physical function. OA progresses slowly over a period of several years; to avoid an exacerbation of symptoms, it is critical to able to diagnose the disease as early as possible. The identification of disease-specific biomarkers may enable such an early diagnosis. The aim of this study was to investigate potential biomarkers of cartilage metabolism in OA using a targeted multiplex approach by single reaction monitoring. Intact looking cartilage of femoral heads from patients with OA (n = 9) or femoral neck fractures (n = 12) was examined. Variations and relative quantifications of 35 selected extracellular matrix (ECM) proteins were analyzed using nano-LC coupled to tandem mass spectrometry. Our study showed statistically significantly increased levels of asporin (ASPN), mimecan (MIME), matrilin-3 (MATN3), cartilage intermediate layer protein 2 (CILP-2), collagen VI, collagen II, and collagen III N-propeptide in OA cartilage compared with non-OA cartilage. The other proteins in the protein panel did not appear to be different between the two groups. In conclusion, we identified a number of cartilage matrix proteins which may represent early molecular changes in the OA process and may have potential to predict the development of OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.