Eicosanoids are 20-carbon lipids generated by the oxidation of arachidonic acid that are involved in physiological signaling in virtually all organ systems. Three primary enzymatic pathways are responsible for their synthesis in mammalian cells: lipoxygenase, cyclooxygenase, and cytochrome P450. They signal through receptor-dependent pathways, and their dysregulation is central to numerous pathological states including cancer and inflammation. Recent advances in their detection and analysis using mass spectrometry have made the study of these molecules more accessible to the research community in general. This review focuses on the available methods for the detection and analysis of eicosanoids and aims to act as a guide for those wishing to approach the analysis of eicosanoids for their own research.