Since fatty acids metabolism plays a crucial role in large number of disorders like cardiovascular disease and certain types of cancer, the detection and quantification of these compounds are of great importance. Since the endogenous fatty acids metabolites are of rather low concentration, sensitive and specific analytical methods are required for the reliable quantification of these compounds. HPLC-MS/MS has emerged as one of the main techniques used for fatty acids metabolites quantification. Creative Proteomics has established sensitive, reliable, and accurate LC-MS/MS method for quantification of fatty acids metabolites.