The gas chromatographic properties of four derivatives of fatty acids (FA), namely fatty acid methyl esters, picolinyl esters, N-acyl pyrrolidides and 4,4-dimethyloxazoline derivatives, which contain various structural features (double bonds, branching, hydroxyl group) in their acyl chains have been compared on a low-polarity capillary column with a mass spectrometer as detector. Temperature programming rates yielding the highest resolution were optimized for each derivative by means of computer-assisted column temperature optimization software. Indeed, the Drylab software represents a valuable assistance for estimating the optimum analysis conditions. Time and efforts required for such method development can greatly be reduced. Different parameters (derivatization procedure, total run time, resolution and response factor) are discussed. N-Acyl pyrrolidides and picolinyl esters appear quite powerful for structure elucidation of polyunsaturated FA by GC-MS, and both these types of FA derivatives can be very well separated on a low-polarity phase.