Cardiovascular disease (CVD) is the major cause of death in the Western hemisphere. Oxidative stress is involved in the pathophysiology of cancer, neurodegenerative conditions and CVD. Lipid peroxidation is one of the oxidative modifications possible in biological systems. The isoprostanes are derivatives of one specific lipid, i.e., arachidonic acid, after lipid peroxidation. Several isoprostanes have been identified in biological tissues and fluids, among them 8-iso prostaglandin F2alpha (8-iso-PGF2alpha, 8-epi-PGF2alpha, iPF2alpha-III, 15-F2t-IsoP) and its metabolite, 2,3-dinor-4,5-dihydro-8-iso-PGF2alpha. The isoprostanes are reliable in vivo markers of lipid peroxidation in humans: they are endogenously formed, characteristic in structure, ubiquitous in nature, stable in- and ex vivo and reliably quantitatable. In this Review, different analytical approaches will be discussed including immunologic, chromatographic and spectrometric techniques with the main emphasis on mass spectrometry. Analysis of isoprostanes applying radio immunoassay (RIA), enzyme immunoassay (EIA), high performance-liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-tandem MS), gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS will be exemplified in the field of cardiovascular research. Results from several clinical studies are included indicating the validity of isoprostanes as surrogate parameters of oxidative stress in cardiovascular disease.