RATIONALE Isoprostanes (IsoPs) are a series of prostaglandin (PG)‐like compounds formed non‐enzymatically through free‐radical‐induced peroxidation of arachidonic acid. They are considered as 'gold‐standard' biomarkers for oxidative stress, in general, and lipid peroxidation, in particular. METHODS A new qualitative and quantitative analytical method for the determination of 13 eicosanoids in human urine using solid‐phase extraction (SPE) and ultra‐pressure liquid chromatography coupled to tandem mass spectrometry (UPLC/MS/MS) has been developed. The SPE was optimized by comparison of the extraction efficiency and recoveries of three distinct cartridges: Strata X‐AW, C18 Sep‐Pak, and Oasis HLB. The UPLC/MS/MS approach in the multiple reaction monitoring (MRM) mode was developed using negative electrospray ionization (ESI). RESULTS The validated method provides a high‐throughput assay with an adequate linearity from 0.16 to 330 ng mL –1 . The limit of detection (LOD) and limit of quantification (LOQ) for each analyte showed low intervals (0.021–0.64 ng mL –1 and 0.042–1.28 ng mL –1 , respectively). Urinary IsoPs were determined in 24 healthy volunteers and ranged from 685 to 3480 ng 24 h –1 and from 864 to 7511 ng 24 h –1 in urine from women and men, respectively. CONCLUSIONS This analytical method could constitute a useful tool for the determination of oxidative stress biomarkers in clinical studies in which IsoPs may evidence early pathological conditions, as suggested by the determination of the baseline IsoPs content in human urine, since it improves upon the detection capacity of previously described methods. The quantity of IsoPs excreted in urine was higher than that found in previous reports due to the total hydrolysis of the conjugated forms. Copyright © 2012 John Wiley & Sons, Ltd.