Proteomic analyses of cartilage and, to a lesser extent, of bone have long been impaired because of technical challenges related to their structure and biochemical properties. We have developed a unified method based on phenol extraction, 2DE, silver staining, and subsequent LC-MS/MS. This method proved to be efficient to characterize the proteome of equine cartilage and bone samples collected in vivo. Since proteins from several cellular compartments could be recovered, our procedure is mainly suitable for in situ molecular physiology studies focused on the cellular content of chondrocytes, osteoblasts, and osteoclasts as well as that of the extracellular matrix, with the exception of proteoglycans. Our method alleviates some drawbacks of cell culture that can mask physiological differences, as well as reduced reproducibility due to fractionation. Proteomic comparative studies between cartilage and bone samples from healthy and affected animals were thus achieved successfully. This achievement will contribute to increasing knowledge on the molecular mechanisms underlying the physiopathology of numerous osteoarticular diseases in horses and in humans.