Isoprostanes are formed after peroxidation of arachidonic acid and are promising biomarkers for reactive oxygen species. A LC–MS/MS based method was developed for the quantitation of two isoprostanes (iPF 2α -III and 8,12-iso-iPF 2α -VI) in hepatocytes, tissue and urine samples of rats. A column switching method was used to reduce sample preparation to a minimum. Precision was 9.4% and accuracy was between 96 and 114% for free iPF 2α -III in tissue at concentrations from 1.9 to 6.1ng/g. Treatment of rats with CCl 4 to induce oxidative stress resulted in a dose-dependent increase (two- to three-fold) of iPF 2α -III and 8,12-iso-iPF 2α -VI in liver and kidney. For both isoprostanes an increase of four- to five-fold was observed in CCl 4 treated hepatocytes and six- to eight-fold in CCl 4 treated and glutathione depleted hepatocytes. In conclusion, the presented method is sensitive, specific and precise to be applied for the quantitation of iPF 2α -III and 8,12-iso-iPF 2α -VI which are shown to increase by CCl 4 treatment in vitro and in vivo.