A new derivatization procedure for fatty acids followed by HPLC analysis with UV detection is described. Originally, derivatization was carried out with acetone solutions of 2,4'-dibromoacetophenone and triethylamine at 50°C for 2h. To prevent oxidation and isomerization, unsaturated fatty acids and conjugated dienes in particular were derivatized at a lower temperature (40°C) for 30 min using a concentrated solution of 2,4'-dibromoacetophenone in acetone and triethylamine. The new derivatization method decreases the risk of degradation and isomerization of unsaturated fatty acids. Due to the use of higher concentrations of substrates in reaction mixtures, significantly enhanced sensitivity of fatty acid analysis is observed. In contrast to the original derivatization method, the new one can be successfully used for both large and trace concentrations of fatty acids in various types of biological materials.