Progress in the knowledge of pathogenic mechanisms and a better definition of the disease, together with the availability of new technologies, have recently improved the value of laboratory investigations in osteoarthritis (OA). The main objectives of these findings are early diagnosis, assessment of disease activity and severity, and evaluation of therapeutic effects. In this context, biochemical markers are potentially useful, as they are non-invasive and non-expansive. However, among the numerous substances increasingly proposed for these purposes, very few may be considered as true disease markers in OA; COMP, antigenic keratan sulphate, hyaluronic acid, YKL-40, type III collagen N-propeptide and urinary glucosyl-galactosyl pyridinoline seem to be the most promising. However, serum or urinary determinations of these molecules are difficult to interpret adequately, due to their complex metabolism. Careful analysis of synovial fluid, mainly directed to leukocyte count and crystal detection, is still essential for diagnosis, but also for the evaluation of the levels of important markers of local inflammation, such as metalloproteinases and cytokines, which seem to be crucial in the pathogenesis of OA.