Although the cytotoxic effects of ethanol and dimethyl sulfoxide (DMSO) on some cell lines have been shown in the literature, there is no study about the cytotoxic effects of these solvents on common used human cell lines, such as melanoma (VMM917), lung cancer (A549), colon cancer (WiDr), normal colon (CCD 841 CoN) and fibroblast (BJ) cells. The aim of this study was to determine the cytotoxic effects of ethanol and DMSO on human breast (MCF-7), liver (HepG2) and cervix (HeLa) cancer cells in addition to above mentioned cells. For this purpose, the cells were treated with different concentrations (0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1%, 2%, and 4%, v/v) of ethanol and DMSO and then subjected to MTT assay. According to the results, ethanol and DMSO exhibited dose-dependent cytotoxicity in all cells studied. The most DMSO and ethanol sensitive cells were WiDr and VMM917, while the most resistant cells were determined as BJ and A549, respectively. The results revealed that the concentration range in which ethanol and DMSO exhibited cytotoxic effect in each cell line is different. In order not to cause false positive and negative results, the concentration range in which the solvents used in cell culture studies do not have cytotoxic effects should be determined.