Fatty acids are the building blocks of fats. They are chemically composed of carboxylic acids with long aliphatic chains and can be branched, unbranched, saturated, or unsaturated. Fatty acids, especially the unsaturated, perform several biological functions and possess various health benefits. Therefore, a strong focus is observed on investigating fatty acids, specifically those that could have biomedical applications. This chapter discusses the traditional and modern extraction methods of fatty acids like microwave-assisted technique, supercritical fluid extraction, and ultrasound extraction. The methods of quantification of fatty acids like thin layer chromatography, gas chromatography, high performance liquid chromatography, supercritical fluid chromatography, mass spectrometry, and capillary electrophoresis have also been discussed.