Lipid mediators derived from essential fatty acids, such as arachidonic acid, play important roles in physiologic and pathophysiologic processes. Prostaglandins, thromboxane, and leukotrienes are well‐known eicosanoids that play critical roles in hemodynamics and inflammation. New families of mediators were recently uncovered that constitute a new genus stimulating resolution of acute inflammation, and are organ‐protective. These include the resolvins (E‐series and D‐series), protectins (neuroprotectin D1/protectin D1), and maresins biosynthesized from omega‐3 essential fatty acids. Phagocytes play major roles in tissue homeostasis and have a high capacity to produce these mediators, which depend on their tissue and state of activation. It is important to select appropriate methods for identifying target mediators and pathway biomarkers. In this unit, we review state‐of‐the‐art approaches to identify and profile eicosanoid and docosanoid pathways, including specialized pro‐resolving mediators resolvins, protectins, and maresins, in relation to their biosynthesis and inactivation by neutrophils and macrophages. Curr. Protoc. Immunol. 95:14.26.1‐14.26.26. © 2011 by John Wiley & Sons, Inc.