Oxidative stress is implicated as one of the major underlying mechanisms in a variety of human diseases. Reactive radicals, derived primarily from molecular oxygen, readily attack a variety of critical biological molecules, including DNA, cellular proteins, and lipids. Since lipid peroxidation is a central feature of cerebral oxidant injury, measurements of peroxidation products, such as F2-isoprostanes (F2-IsoPs) and F 4-neuroprostanes (F4-NeuroPs), represent the most accurate approaches to identify oxidative injury in vivo. Our laboratory uses a gas chromatography/mass spectrometry negative ion chemical ionization with select ion monitoring for quantification of these biomarkers of oxidative stress in a plethora of biological media. This review summarizes state-of-the-art methodology for F2-IsoPs and F4-NeuroPs analyses and discusses the utility of these prostaglandin-like compounds as in vivo biomarkers of neuronal oxidative injury.