Background Vitrification is a method of cryopreservation by which cells and tissues can be preserved at low temperatures using cryoprotective agents (CPAs) at high concentrations (typically ⩾6.0 M) to limit the harmful effects of ice crystals that can form during cooling processes. However, at these concentrations CPAs are significantly cytotoxic and an understanding of their toxicity characteristics and interactions is important. Therefore, single-CPA and multiple-CPA solutions were evaluated for their direct and indirect toxicities on chondrocytes. Methods Chondrocytes were isolated from human articular cartilage samples and exposed to various single-CPA and multiple-CPA solutions of five common CPAs (dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), glycerol (Gy) and formamide (Fm)) at both 6.0 and 8.1 M concentrations at 0 °C for 30 min. Chondrocyte survival was determined using a fluorescent cell membrane integrity assay. The data obtained was statistically analyzed and regression coefficients were used to represent the indirect toxicity effect which a specific combination of CPAs exerted on the final solution’s toxicity. Results Multiple-CPA solutions were significantly less toxic than single-CPA solutions (P < 0.01). The indirect toxicity effects between CPAs were quantifiable using regression analysis. Cell survival rates of approximately 40% were obtained with the four-CPA combination solution DMSO–EG–Gy–Fm. In the multiple-CPA combinations, PG demonstrated the greatest degree of toxicity and its presence within a combination solution negated any benefits of using multiple lower concentration CPAs. Conclusions Multiple-CPA solutions are less cytotoxic than single-CPA solutions of the same total concentration. PG was the most toxic CPA when used in combinations. The highest chondrocyte survival rates were obtained with the 6.0 M DMSO–EG–Gy–Fm combination solution.