This study was performed to standardize a dentin barrier test with the substitute and evaluate the cytotoxicity of one-step self-etching bonding agents. Each of the natural bovine dentin and polyurethane discs were 500-μm thick and were tested using a perfusion device. Following the treatment with 0.05% phenol on the natural bovine disc or three kinds of polyurethane discs—30, 40, and 50 pcf (pounds per cubic foot)—cell viability of L-929 was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and expressed as percentages of non-treated group, respectively. A substitute showing permeability similar to that of bovine dentin was determined based on this result. Cytotoxicity test of bonding agents was performed by the selected substitute, the results of which were expressed as percentages of the control. In addition, SEM images were taken after the tests. The cell viability by 40-pcf polyurethane disc was not statistically different from that by bovine dentin disc (P
> 0.05). Futurabond DC resulted in the highest cell viability and Bond force the lowest by the 40-pcf polyurethane disc (P
< 0.05). The adhesives on the 40-pcf polyurethane disc changed cellular morphology with different degrees on the SEM images. This standardized test might be useful for assessing the cytotoxicity of dental materials applied to dentin before clinical applications.