F2-isoprostanes (F2-isoPs) represent a new family of biomarkers for oxidative stress generated by free radical attack of membrane-bounded arachidonic acid. Esterified F2-isoPs can be found in tissue or plasma lipids whereas the free form F2-isoPs, hydrolyzed by phospholipase, is mainly present in body fluids. The extent of systematic damage due to oxidative stress within the body can be assessed by the determination of plasma or urine F2-isoPs. The determination of F2-isoPs in clinical practice is not often used due to the complexity to extract the compounds from their biologic matrixes before the analysis step. In most of published protocols, extraction procedure is critical and timeconsuming, requiring successive chromatographic steps. Moreover, some of these procedures lead to a substantial loss of target compounds. In order to improve sample preparation steps and final recovery, others methods have been developed and optimized. For detection of F2-isoPs, two main analytical approaches have been adopted. The first one involves immunological methods and the second approach is based on chromatographic separation and detection by mass spectrometry. A large amount of works has been done in the field of isoprostane analysis, but until now, no standardized method seems to emerge. Indeed, described methodologies differ either in the sample preparation steps or in the detection techniques or both. In the present review, the most commonly used methods are presented and compared in terms of extraction, purification, and analysis of F2-isoPs, taking into account the various origins of biological samples.