A number of studies have revealed that oxidative stress plays important roles in the pathogenesis of various diseases, such as cancer, diabetes and atherosclerosis[1,2]. The 8-isoprostane present in biological fluids is produced from arachidonic acid by a non-enzymatic, free radical-catalyzed reaction, and has been proposed as a reliable marker for lipid peroxidation and oxidative stress in vivo. 8-Isoprostane is chemically stable, in contrast to other conventional markers of oxidative stress, and its levels in either plasma or urine are elevated in subjects who smoke[4-8] and ingest alcohol[9,10],as well as in patients with diabetes mellitus, heart disease[12-15], hypertension, preeclampsia and asthma. Urinary 8-isoptostane levels increase during the progression of alcohol-induced liver disease and are decreased by abstinence. However, accurate measurement of 8-isoptostane is not easy and requires special instruments, such as GC/MS or LC/MS, since various types of analogues and metabolites are present in biological fluids. For instance, the plasma and urinary 8-isoprostane levels determined by a recently developed immunoassay were much higher than those obtained by GC/MS or LC/MS assays[19,20]. This could be attributed to cross-reaction of the 8-isoprostane analogues and metabolites in the samples with the 8-isoprostane antibody used in the immunoassay. In this study, we developed a new method for pretreatment before analyzing by the commercially available ELISA kit, and performed various examinations for accurate assay of 8-isoprostane. Using this method, we examined the effects of drinking and smoking habits against the levels of plasma 8-isoprostane in healthy Japanese volunteers.