Topical drug treatment of the eye exposes ocular tissues to a high drug concentration. Genotoxicity assessment in ocular tissues has not been established to date. Therefore, we investigated the in vitro comet assay by incubating cultured human corneal epithelial (HCE-T) cells with known mutagens. The alkaline comet assay was conducted to measure the DNA strand breakage yield. When the cells were incubated with methyl methanesulfonate (MMS) for 1 hr, hydrogen peroxide (H₂O₂) for 15 min and actinomycin D (AMD) for 1 hr, statistically significant increases of percentage (%) DNA in the tail were noted in MS-, H₂O₂-, and AMD-treated cells at 100, 10, and > 10 μM, respectively. When the cells were treated with mitomycin C (MMC) or 5-bromouracil (5-BrU), %DNA in the tail was unchanged even at the highest concentration. Hedgehog cells were found in MMS- and H₂O₂-treated cells at 1000 and > 100 μM, respectively. The response to each compound was consistent with results previously reported in other cells. In conclusion, the in vitro comet assay using HCE-T cells can detect DNA strand breakage induced by mutagens. This method has a possibility to become a conventional screening tool to assess the genotoxicity of drugs applied to ocular surface.