Eicosanoids begin as a single poly-unsaturated fatty acid, arachidonic acid. Yet from this simple origin, hundreds of bioactive signaling molecules can be created, often within minutes of receiving an initiation signal. Many of these bioactive lipids have a functional partner that generates an opposing effect and helps prevent any one pathway from signaling uncontrollably in the body. For this reason, we have adapted liquid chromatography and tandem mass spectrometry into a lipidomic platform that can be employed to study eicosanoid signaling in a comprehensive manner. The overarching goal of such an approach is to elucidate the role of eicosanoids in physiology and disease at the cellular and organismal level, in hopes of identifying better targets for pharmaceutical intervention. This thesis first discusses the pathways of eicosanoid biosynthesis and catabolism, as well as the differences between humans and the model organisms commonly used as research surrogates (mouse and rat). Using this background, a mass spectra library of known eicosanoids was generated and used to create high- throughput lipidomic methodology using tandem mass spectrometry that facilitated several different studies. We investigated a number of receptor-mediated eicosanoid activation pathways in the macrophage cell, identifying two distinct pathways that synergistically produce eicosanoids in combination. Using this study, we turned the macrophage model into a tool for developing potent and selective inhibitors of inflammatory metalloproteins. At the organismal level, we developed a model of inflammation in mice infected with Lyme disease that integrates the major eicosanoid pathways, from initial infection to resolution of inflammation