Eicosanoids constitute a large class of biologically active arachidonic acid (AA) metabolites that play important roles in numerous physiological processes. Eicosanoids are produced by several distinct routes, including the cyclooxygenase, lipoxygenase, and P450 enzymatic pathways, as well as by nonenzymatic processes. In order to completely understand the eicosanoid response of a cell or tissue to a given stimulus, measuring the complete profile of eicosanoids produced is important. Since the eicosanoids are products of a single species, AA, and represent, for the most part, the addition of various oxygen species, the hundreds of eicosanoids have very similar structures, chemistries, and physical properties. The identification and quantitation of all eicosanoids in a single biological sample are a challenging task, one that high-performance liquid chromatography-mass spectrometry (LC-MS) is well suited to handle. We have developed a LC-MS/MS procedure for isolating, identifying, and quantitating a broad spectrum of eicosanoids in a single biological sample. We currently can measure over 60 eicosanoids in a 16-min LC-MS/MS analysis. Our method employs stable isotope dilution internal standards to quantitate these specific eicosanoids. In the course of setting up the LC-MS system, we have established a library that includes relative chromatographic retention times and tandem mass spectrometry data for the most common eicosanoids. This library is available to the scientific community on the website www.lipidmaps.org.