Oxidant stress has been implicated in a wide variety of disease processes. One method to quantify oxidative injury is to measure lipid peroxidation. Quantification of a group of prostaglandin F(2)-like compounds derived from the nonezymatic oxidation of arachidonic acid, termed the F(2)-isoprostanes (F(2)-IsoPs), provides an accurate assessment of oxidative stress both in vitro and in vivo. In fact, in a recent National Institutes of Health-sponsored independent study, F(2)-IsoPs were shown to be the most reliable index of in vivo oxidant stress when compared against other well-known biomarkers. This article summarizes current methodology used to quantify these molecules. Our laboratory's method to measure F(2)-IsoPs in biological fluids and tissues using gas chromatography-mass spectrometry is detailed herein. In addition, other mass spectrometric approaches, as well as immunological methods to measure these compounds, are discussed. Finally, the utility of these molecules as in vivo biomarkers of oxidative stress is summarized.