The methods described in this chapter are designed for rapid and convenient separation and quantitation of calpain isozymes in cultured osteoblastic cells by modern semiautomated strong-anion exchange FPLC. The isozymes of the calpain-calpastatin system are abundant in osteoblastic or bone-forming cells, where they are regulated by parathyroid hormone (1) and bone morphogenetic protein (2). High levels of calpain II immunoreactivity are present in other mineralizing tissues, including calcifying cartilage (3) and fracture callus (4). Extracellular calpain II is present in terminally differentiated chondrocyte cultures, where it degrades large proteoglycan monomers and contributes to the initiation of biomineralization (5). Calpain protein and activity are present in osteoarthritic synovial fluid and synoviocytes (6), where calpain catalyzes degradation of major proteoglycan core proteins, abolishing hyaluronic acid binding and damaging cartilage (7).