To establish a simple screening system for estimating efficacy of an agent for an oxidative-related lesion, we investigated the damage in isolated rat hepatocytes exposed to 75 μM tert-butyl hydroperoxide (t-BuOOH) and then subsequently incubated the cells in fresh medium. By electron spin resonance spectroscopy analysis using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), DMPO adducts of tert-butoxyl radicals and carbon center radicals were detected during the t-BuOOH exposure, and DMPO-OH formation was detected after t-BuOOH removal. In t-BuOOH-exposed cells, the level of phosphatidylcholine hydroperoxide (PCOOH), a peroxidative product of biomembranes in the hepatocytes, and the leakage of enzymes into the culture medium were significantly increased. An increase in acid phosphatase (AP) activity representing lysosome destabilization preceded the aspartate oxoglutarate aminotransferase (AST), alanine oxoglutarate aminotransferase (ALT) and lactate dehydrogenase (LDH) leakage. Ninjin-yoei-to added to the culture medium following the t-BuOOH exposure significantly inhibited the PCOOH formation and the leakage of AP, AST, ALT and LDH, concentration-dependently. Ninjin-yoei-to at 1 mg/ml in culture medium completely diminished these increases in enzyme activities down to the background levels found in control experiments and this reduction was greater than the most effective α-tocopherol concentration of 20 μmol/ml. Considering all of these results, it is likely Ninjin-yoei-to may exert its protective effect by anti-oxidative action and membrane stabilization.