To characterize the in vitro toxicity of vancomycin on articular cartilage. Osteochondral samples extracted from fresh, juvenile porcine stifle (knee) joints were exposed to vancomycin saline concentrations of 2, 5, and 10 mg/mL and compared with a saline and nonsaline treated control. Comparison of chondrocyte viability was assessed with histology examination of chondrocyte degeneration using the Mankin criteria and a live/dead staining using ethidium homodimer-2 and calcien acetoxymethylester stain and confocal laser scanning microscopy. A comparison of chondrocyte viability in the control sample and 2 mg/mL group showed no statistical difference (P = 0.38). The chondrocyte toxicity was statistically significantly higher in both the 5 mg/mL (P = 0.003) and 10 mg/mL (P < 0.001) experimental groups in comparison to the control sample. Chondrocyte death increased in a statistically significant dose-dependent fashion. Histologically, the Mankin scores were higher for the saline-treated group in comparison to the untreated control group. Vancomycin is toxic to articular chondrocytes in concentrations of 5 mg/mL and greater. Strategies for controlling concentration must be developed before routine application of topical antibiotics around synovial joints is performed.