Matrix metalloproteinases (MMPs) play crucial roles in tissue homeostasis and pathologies by remodeling the extracellular matrix. Previous studies have demonstrated the biological activities of MMP-derived cleavage products. Furthermore, specific fragments can serve as biomarkers. Therefore, an in vitro cleavage assay to identify substrates and characterize cleavage patterns could provide important insight in disease-relevant mechanisms and the identification of novel biomarkers. In the pathogenesis of osteoarthritis (OA), MMP-2, -8, -9 and -13 are of vital importance. However, it is unclear which protease can cleave which matrix component. To address this question, we established an in vitro cleavage assay using recombinantly expressed MMPs and the two cartilage matrix components, COMP and thrombospondin-4. We found a time- and concentration-dependent degradation and an MMP-specific cleavage pattern for both proteins. Cleavage products can now be enriched and purified to investigate their biological activity. To verify the in vivo relevance, we compared the in vitro cleavage patterns with serum and synovial fluid from OA patients and could indeed detect fragments of similar size in the human samples. The cleavage assay can be adapted to other MMPs and substrates, making it a valuable tool for many research fields.