Most lipids are best characterized by their fatty acids which differ in chain length, the degree of unsaturation, configuration and position of the double bonds, and the presence of other functionalities. The fatty acid profiles are currently most frequently determined by capillary gas chromatography of the methyl esters which are prepared by a variety of methods. These are discussed with an emphasis on more recent developments, along with the stationary phases used for the separations and the methods employed for identification of the fatty acids. HPLC is applied less frequently for ascertaining fatty acid profiles than GC, but a very large number of derivatives for ultraviolet and fluorescence detection have been proposed. This method continues to evoke increasing interest, particularly in conjunction with fluorescence detection. This technique enables attainment of greater sensitivities than with standard GC methods employing flame ionization detection. Extensive applications of it to the analysis of free fatty acids in blood and other biomedical samples are clearly discernible. Other methods, including supercritical fluid chromatography, have found only limited application for fatty acid profiling.