Four techniques, i.e., gas-liquid chromatography, gas-liquid chromatography + thin-layer chromatography, and two spectroscopic methods, Fourier transform infrared spectroscopy and optothermal window, a variant of the open photoacoustic cell, were intercompared to determine their potential to detect the total trans fatty acid content in margarine. At the same time, this study represents a first application of the optothermal window technique at long wavelengths (10 μm). The total trans fatty acid data obtained by different methods show good mutual agreement. Besides offering several attractive advantages above conventional methods, the optothermal window also proved suitable for measuring total trans fatty acid content as low as 2%.