Abstract: PR assay, YR assay and CPE inhibition assay were used to determine the effect of Tai-Ding-An (3-phthalimido-2-oxo-butyraldehyde-bis-thiosemicarbazone, V 6133) against HSV-1 and HSV-2 in vitro. The results of PR assay showed that the ratio of the plaque number was near to zero in the presence of drug concentrations of more than 50μg/ml in comparison with DMSO control. Dose-response curve obtained by YR assay revealed that Tai-Ding-An markedly inhibited replication of both HSV-1 and HSV-2. PRK cells were treated with 100 μg/ml of Tai-Ding-An and incubated for 2 days, no HSV-induced CPE was observed while CPE was very evident in the control. No CPE was observed for up to 5 days when the PRK cells were treated continuously with the media containing 100 μg/ml of the drug and changed daily. From these results it would appear that Tai-Ding-An is an effective agent against HSV-1 and HSV-2.In vitro experiments also showed that the toxic effect was mild when the concentration of Tai-Ding-An was less than 100 μg/ml.