Misregulation of oxidative and antioxidative processes in the organism – oxidative stress – contributes to the pathogenesis of different diseases, e.g. inflammatory or neurodegenerative diseases. Oxidative stress leads to autoxidation of polyunsaturated fatty acids giving rise to prostaglandin-like isoprostanes (IsoP) and isofurans (IsoF). On the one hand they could serve as biomarker of oxidative stress and on the other hand may act as lipid mediators, similarly as the enzymatically formed oxylipins. In the present paper we describe the development of an LC-ESI(-)-MS/MS method allowing the parallel quantification of 27 IsoP and 8 IsoF derived from 6 different PUFA (ALA, ARA, EPA, AdA, n6-DPA, DHA) within 12 min. The chromatographic separation was carried out on an RP-C18 column (2.1 × 150 mm, 1.8 μm) yielding narrow peaks with an average width at half maximum of 3.3–4.2 s. Detection was carried out on a triple quadrupole mass spectrometer operating in selected reaction monitoring mode allowing the selective detection of regioisomers. The limit of detection ranged between 0.1 and 1 nM allowing in combination with solid phase extraction the detection of IsoP and IsoF at subnanomolar concentrations in biological samples. The method was validated for human plasma showing high accuracy and precision. Application of the approach on the investigation of oxidative stress in cultured cells indicated a distinct pattern of IsoP and IsoF in response to reactive oxygen species which warrants further investigation. The described method is not only the most comprehensive approach for the simultaneous quantification of IsoP and IsoF, but it was also integrated in a targeted metabolomics method (Ostermann et al. (2015) Anal Bioanal Chem) allowing the quantification of in total 164 oxylipins formed enzymatically and non-enzymatically within 30.5 min.