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Background The cytotoxicity of dermal substitutes may be increased by the very processes used to deplete the cells. The present research aimed to investigate the method for monitoring the cytotoxicity of cell-free dermal substitutes using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method.Methods The cytotoxicity of four dermal substitutes was evaluated using the MTT method according to the standards set by the Chinese State Food and Drug Administration (SFDA). Swine acellular dermal matrix (SADM) and goat acellular dermal matrix (GADM) were produced using a repeated freeze-thaw method. Human dermal matrix glutaraldehyde composite (HADM-G) and SADM cross-linked with glutaraldehyde (SADM-G) were produced using conventional methods. Results The cytotoxicity of all dermal substitutes ranged from Grade 0 to Grade 1, meeting the standards of the Chinese FDA. The OD_(490) of both SADM and GADM was higher than that of either HADM-G or SADM-G (P<0.05). Conclusion Dermal substitutes produced by the freeze-thaw method are less cytotoxic than those produced using conventional methods.