Eicosanoids derived from arachidonic acid through cyclooxygenase (COX), lipoxygenase (LOX), and P450 pathways include prostanoids, hydroxyeicosatetraenoic acids (HETEs), leukotrienes (LTs), and epoxyeicosatrienoic acids (EETs). These bioactive lipids play an important role in regulating cell proliferation, apoptosis, tissue repair, blood clotting, blood vessel permeability, inflammation, and immune cell behavior. Moreover, some of these eicosanoids also modulate inflammation and tumor growth in cancer tissues, and may serve as biomarkers for monitoring colorectal cancer progression or as an intermediate marker for the pharmacologic activity of chemopreventive agents. Development of sensitive, rapid, and specific methods for determining eicosanoid levels accurately will facilitate an understanding of the biologic functions of these lipid mediators and will broaden our insight of the importance of these bioactive lipids in vivo. However, quantitative determination of eicosanoids in biological samples has presented a problem to many investigators. It is necessary to understand the advantages and limitations of each method for quantitative analysis of specific eicosanoids in various types of biological samples. Here we evaluate the methodology of the measurement of eicosanoids in biological samples.