Fatty acids are a concentrated source of energy, are necessary components of cell walls, and possess several physiological functions like gene regulation and biological signalling. The role of fatty acids in healthy fetal development, prevention of cardiovascular diseases, and Alzheimer is also observed. The ester of some fatty acids is used as food flavorings. They are also used as inactive ingredients in drug preparation. These applications of fatty acids have increased their demand in the purified form. To isolate fatty acids, the fats and oils are first saponified to extract glycerol and produce free fatty acids. These are further isolated by urea fractionation, low temperature crystallization, molecular distillation, chromatographic separation, enzymatic purification, and supercritical extraction. A combination of purification methods produces fatty acids with high purity. The isolated fatty acids can be further characterized using chromatographic techniques like thin layer chromatography, gas chromatography, high pressure liquid chromatography, and mass spectrometry.