Diagnosing osteoarthritis (OA) in dogs can be problematic due to the lack of early detection methods. Radiographic changes used to diagnose OA occur only in later stages of the disease. Inflammatory and degenerative biomarkers (eg, tumor necrosis factor alpha, interleukin-1 beta [IL-1 beta], tenascin-c [TN-C], matrix metalloproteinase-2 [MMP-2]) have been correlated with canine joint inflammation, and immunoassays are commercially available. Glucose, pH, and lactate metabolic biomarkers also increase in the synovial fluid of joints with OA, and tests for these biomarkers are simpler and less expensive. This pilot study of dogs with OA found that metabolic markers, pH, and glucose are significantly increased in OA-affected joints as compared with normal joints. They also found that synovial fluid lactate was significantly decreased in affected joints. Of the proinflammatory biomarkers, IL-1 beta, TN-C, and MMP-2 were significantly increased in OA-affected joints. None of the values correlated to radiographic findings, likely due to the difficulty of radiographically detecting OA in early stages of disease; correlations with MRI findings should be studied.