The eicosanoids are a large family of arachidonic acid oxidation products that contain 20 carbon atoms. Cyclooxygenase (COX)-derived eicosanoids have important roles as autacoids involved in the regulation of cardiovascular function and tumor progression. Lipoxygenase (LO)-derived eicosanoids have been implicated as important mediators of inflammation, asthma, cardiovascular disease and cancer. Cytochrome P-450 (P450)-derived eicosanoids are both vasodilators and vasoconstrictors. There is intense interest in the analysis of reactive oxygen species (ROS)-derived isoprostanes (isoPs) because of their utility as biomarkers of oxidative stress. Enzymatic pathways of eicosanoid formation are regioselective and enantioselective, whereas ROS-mediated eicosanoid formation proceeds with no stereoselectivity. Many of the eicosanoids are also present in only pM concentrations in biological fluids. This presents a formidable analytical challenge because methodology is required that can separate enantiomers and diastereomers with high sensitivity and specificity. However, the discovery of atmospheric pressure ionization (API)/MS methodology of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and electron capture (EC) APCI has revolutionized our ability to analyze endogenous eicosanoids. LC separations of eicosanoids can now be readily coupled with API ionization, collision induced dissociation (CID) and tandem MS (MS/MS). This makes it possible to efficiently conduct targeted eicosanoid analyses using LC-multiple reaction motoring (MRM)/MS. Several examples of targeted eicosanoid lipid analysis using conventional LC-ESI/MS have been discussed and some new data on the analysis of eicosanoids using chiral LC-ECAPCI/MS has been presented.