Prostaglandins, thromboxane, leukotrienes, isoprostanes and other arachidonic acid metabolites are structurally closely related, potent, biologically active compounds. One of the most challenging tasks in eicosanoids research has been to define the role of the various eicosanoids in human health and disease, and to monitor the effects of drugs on the in vivo synthesis of these lipid mediators in man. Great advances in instrumentation and ionization techniques, in particular the development of tandem mass spectrometry and negative-ion chemical ionization (NICI), in gas chromatography and also advances in methodologies for solid-phase extraction and sample purification by thin-layer chromatography and high-performance liquid chromatography have been made. Now gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS in the NICI mode are currently indispensable analytical tools for reliable routine quantitation of eicosanoid formation in vivo in humans. In this article analytical methods for eicosanoids based on GC-MS and GC-tandem MS are reviewed emphasizing the quantitative measurement of specific index metabolites in human urine and its importance in clinical studies in man. Aspects of method validation and quality control are also discussed.