Fatty acids consist of straight chains with an even number of carbon atoms with a carboxyl group. They build lipids, are a key component in cell membranes and participate in the transmission of substances and signals in the cells of living beings. Fatty acids can be divided according to saturation. Saturated fatty acids contain only single bonds between carbon atoms, while unsaturated fatty acids have one or more double bonds. Polyunsaturated fatty acids that the human body cannot synthesize on its own are called essential fatty acids. These are linoleic, linolenic and arachidonic fatty acids and must be consumed with food. From essential fatty acids, series of omega fatty acids are created in the body, which are extremely important for the normal functioning of the body. Fatty acids are determined in the form of their methyl esters, and for this purpose a mixture of 37 methyl esters of fatty acids was used as a standard. The method for separation and qualitative and quantitative determination was developed using gas chromatography with a flame ionization detector (GC-FID). The method recommended by the column manufacturer was modified and optimized by changing the temperature program to successfully separate 37 fatty acid esters. The examined parameters of the evaluation of the method (range, linearity, limit of proof and determination, accuracy, repeatability, mean precision and suitability of the system) proved to be satisfactory. The method was also applied to edible oil samples and fatty acid standards. The results of real samples indicated an important step of sample preparation and it was observed that it is necessary to improve the step of esterification of fatty acids in oils.