Osteoarthritis (OA) is the most frequent rheumatic disease and is the leading cause of disability in developed countries. Its poorly understood pathophysiology limits the discovery of targets for pharmacological intervention and there are few effective medical treatments beyond pain control and surgery. Proteomic technologies may help identifying new targets of OA as well as diagnostic or therapeutic biomarkers, which has stimulated interest in this field. In this review, we discuss the most recent findings arising from the use of proteomics for the identification of OA biomarkers in synovial fluid and serum, and for the discovery of possible therapeutic targets in cartilage or by using chondrocyte culture systems.