Eicosanoids, arachidonic acid-derived signaling lipid mediators, are newly formed and nonstorable molecules that have important roles in physiological and pathological processes. EicosaCell is a microscopic assay that enables the intracellular detection and localization of eicosanoid lipid mediator-synthesizing compartments by means of a strategy to covalently cross-link and immobilize eicosanoids at their sites of synthesis followed by immunofluorescent-based localization of the targeted eicosanoid. EicosaCell is a versatile assay which allows analyses of different types of cell preparations, such as cells isolated from humans or harvested cells from in vivo models of inflammation and adherent or suspension cells stimulated in vitro. EicosaCell assays have been successfully used to identify different intracellular compartments of synthesis of prostaglandins and leukotrienes upon cellular activation. This is of particular interest given that over the past decade intracellular compartmentalization of eicosanoid-synthetic machinery has emerged both as a key component in the regulation of eicosanoid synthesis and in delineating functional intracellular and extracellular actions of eicosanoids. This review covers basics of EicosaCell assay including its selection of reagents, immunodetection design as well as some troubleshooting recommendations.