A sample preparation method based on the simultaneous microwave-assisted extraction–esterification (SMAEE) was developed for the determination of the fatty acid composition of foodstuffs by gas chromatography–mass spectrometry. The proposed sample preparation method was validated by comparison with the reference Soxhlet extraction method followed by derivatisation by ester formation and the same determination step. The fatty acid compositions and the extraction efficiencies obtained using the proposed SMAEE method and the reference method was statistically similar. The results showed that compared to the conventional method, the SMAEE method offered the advantages of short sample preparation time, low consumption of expensive organic solvents and lower energy consumption. This good agreement between results provided by both the SMAEE and the reference method demonstrates the usefulness of the former as a routine method for the treatment of food samples prior to trans fatty analysis.