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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\u2010specific 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 (<jats:italic>n<\/jats:italic>\u2009=\u20099) or femoral neck fractures (<jats:italic>n<\/jats:italic>\u2009=\u200912) was examined. Variations and relative quantifications of 35 selected extracellular matrix (ECM) proteins were analyzed using nano\u2010LC coupled to tandem mass spectrometry. Our study showed statistically significantly increased levels of asporin (ASPN), mimecan (MIME), matrilin\u20103 (MATN3), cartilage intermediate layer protein 2 (CILP\u20102), collagen VI, collagen II, and collagen III N\u2010propeptide in OA cartilage compared with non\u2010OA 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. \u00a9 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. 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