Free radical-induced peroxidation of arachidonic acid and other polyunsaturated fatty acids esterified to lipids and subsequent hydrolysis generates prostaglandin-like compounds including the isoprostanes and neuroprostanes. These compounds are endogenously formed, characteristic in structure, stable, and accessible to quantitative determination in tissue, plasma and urine. Isoprostanes have emerged as reliable markers of lipid peroxidation in vivo in humans. Among them 8-iso-prostaglandin (PG) F2α (8-iso-PGF2α, 8-epi-PGF2α, 15-F2t-IsoP, iPF2α-III) and its major urinary metabolites, i.e. 2,3-dinor-4,5-dihydro-8-iso-PGF2α and 2,3-dinor-8-iso-PGF2α, are subject of extensive investigation. Different analytical approaches are currently used to isolate, identify and quantify various members of the isoprostane and neuroprostane families. They include chromatographic approaches such as thin-layer chromatography (TLC), highperformance liquid chromatography (HPLC), and gas chromatography (GC), and in particular mass spectrometry (MS)- based techniques. Gas chromatography-mass spectrometry (i.e. GC-MS and GC-tandem MS), previously proved as the most reliable quantitative technique in the field of prostanoid research, was the first method which was applied to measure isoprostanes. In recent years, liquid chromatography-tandem mass spectrometry (i.e. LC-tandem MS) has also been introduced and established in the analysis of isoprostanes and related compounds. The present paper gives an overview of analytical methods currently applied to analyze isoprostanes in various biological matrices. Special emphasis is given to the quantitative determination in human urine which is a non-invasive method. In this context, the impact of solid-phase extraction (SPE), immunoaffinity column chromatography (IAC), HPLC, and TLC in sample preparation/purification is discussed. Differences in sample preparation depending on the biological matrix, i.e. tissue, blood plasma, and urine, are outlined. Furthermore, to meet increasing interest in isoprostanes as valid and stable markers of oxidative stress in human disease, results from various clinical trials in the cardiovascular field are discussed and relevant experimental findings as well as potential direct effects of isoprostanes in these pathophysiological settings are reviewed. Moreover, relevant clinical data from other disease entities is summarized in a schematic fashion.