Most critical instruments are not designed for heat sterilization and autoclaving. These items are usually treated with chemical agents such as peracetic acid(PAA), glutaraldehyde (GA) and ortho-phthalaldehyde (OPA). MTT assay is often used to evaluate the in vitro cytotoxicity of these chemical agents. In this study, disinfectants were allowed to come in direct contact with cells. Their cytotoxicity was evaluated based on cell viability and adhesive properties. The results obtained from the direct contact method were compared with those obtained from the conventional MTT assay wherein the disinfectants were added into a nutrient medium. It was found that the two methods yielded very different results, especially when aldehyde- and halogen-containing disinfectants were tested, and that toxicity may be underestimated in the MTT assay. Hence, it can be assumed that the direct contact assay is more accurate when evaluating the cytotoxicity of residual chemicals. It was also observed that the cytotoxicity of PAA was lower than that of GA and OPA.