The protective effect of panduratin A, isolated from Kaempferia pandurata ROXB. (Zingiberaceae), against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity was investigated in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) was used to monitor cytotoxicity. Lipid peroxidation [malondialdehyde (MDA) formation] and intracellular glutathione level were estimated by fluorometric methods. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA). Panduratin A significantly reduced the cell growth inhibition caused by t-BHP. Furthermore, panduratin A ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation, and attenuated glutathione (GSH) depletion in a dose-dependent manner. It was also found that panduratin A reduced intracellular ROS formation caused by t-BHP. These results strongly suggest that panduratin A has significant protective ability against oxidative damage caused by reactive intermediates.