Derivatization of fatty acids is usually carried out with 2,4'-dibromoacetophenone in the presence of excess of basic species at 50 °C for 2 h. Under such conditions, some unsaturated fatty acids can be oxidized or isomerized. To prevent oxidation or isomerization, polyunsaturated fatty acids, particularly conjugated dienes, should be derivatized at ~ -26°C for 6.5 h. The proposed new procedure assures a decrease of degradation or isomerization of unsaturated fatty acids. This procedure provides a simple method for preparation and derivatization of fatty acids in milk, meat, fat and intestinal digesta followed by HPLC analysis with UV detection.