Evaluation of the 50% inhibitory concentration (IC50) of acetylsalicylic acid, ferrous sulfate, amitriptyline, methanol, isopropanol and ethylene glycol was done on human cancer cells cultured in in vitro conditions. Three different in vitro assays were used in this study: the plating efficiency test, the microprotein test and the neutral red uptake test. Obtained results were evaluated by statistical methods. All used methods seem to be useful for screening a cytotoxic potential of the tested chemicals. The knowledge of cytotoxic effects of frequently used chemicals on mammalian cells is important not only for necessary in vitro genotoxicity and carcinogenicity studies but also for assessing the toxicity of chemicals to find out possible hazards to the human health. Results presented in this paper underline the usefulness of the wider methodological approach for the comparison of the different endpoints as well as a necessity for selection of a battery of in vitro cytotoxicity tests allowing to estimate the possible harmful effects of xenobiotics.