Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and chondroclasts. The present study investigated changes in TRAP activity after chondrocyte death and cartilage damage, and also evaluated the possible use of TRAP as a diagnostic factor in a model of osteoarthritis. We induced experimental osteoarthritis in beagle dogs and separated chondrocytes from articular cartilage using an enzyme probe. Chondrocyte death was induced by proteasome inhibition and TRAIL treatment, and levels of lactate dehydrogenase, reactive oxygen species (ROS), caspase activation and TRAP activity were measured in the chondrocytes and synovial fluid. Proteasome inhibition and TRAIL treatment significantly enhanced chondrocyte death via caspase-8 activation and ROS generation in the primary cultured canine chondrocytes. TRAP activity was highly increased in damaged chondrocytes, but was decreased by blocking chondrocyte death using caspase inhibition or an ROS scavenger. In the synovial fluid of osteoarthritic dogs, TRAP activity as well as caspase activation and ROS levels were higher than those in the normal joint. Our study demonstrated that TRAP is activated by apoptosis and oxidative stress in primary cultured chondrocytes and osteoarthritic joints and also suggests that TRAP may be used as a diagnostic biomarker for detection of cartilage-related diseases, including osteoarthritis.