Human mesenchymal stem cells (hMSCs), residing in bone marrow as well as in the synovial lining of joints, can be triggered to differentiate toward chondrocytes. Thus, hMSCs harbor great therapeutic potential for the repair of cartilage defects in osteoarthritis (OA) and other articular diseases. However, the molecular mechanisms underlying the chondrogenesis process are still in part unknown. In this work, we applied for the first time the stable isotope labeling by amino acids in cell culture (SILAC) technique for the quantitative analysis of protein modulation during the chondrogenic differentiation process of hMSCs. First, we have standardized the metabolic labeling procedure on MSCs isolated from bone marrow (hBMSCs), and we have assessed the quality of chondrogenesis taking place in these conditions. Then, chondrogenic differentiation was induced on these labeled cells, and a quantitative proteomics approach has been followed to evaluate protein changes between two differentiation days. With this strategy, we could identify 622 different proteins by LC-MALDI-TOF/TOF analysis and find 65 proteins whose abundance was significantly modulated between day 2 and day 14 of chondrogenesis. Immunohistochemistry analyses were performed to verify the changes on a panel of six proteins that play different biological roles in the cell: fibronectin, gelsolin, vimentin, alpha-ATPase, mitochondrial superoxide dismutase, and cyclophilin A. All of these proteins were increased at day 14 compared to day 2 of chondrogenic induction, thus being markers of the enhanced extracellular matrix synthesis, cell adhesion, metabolism, and response to stress processes that take place in the early steps of chondrogenesis. Our strategy has allowed an additional insight into both specific protein function and the mechanisms of chondrogenesis and has provided a panel of protein markers of this differentiation process in hBMSCs.