In 2006, the US FDA mandated the declaration of the total trans fat content on the Nutrition Fact label of foods including dietary supplements when a product contained 0.5 or more grams of trans fatty acid per serving; the minimum corresponding trans fat content is estimated to be approximately 2% of total fat. The FDA definition is based on chemical structure and includes only fatty acids with one or more isolated double bonds in the trans configuration. Several issues negatively impacted the sensitivity of the current official infrared (IR) methods, thus limited the quantitation of trans fat to 5% of total fat. To improve sensitivity and accuracy and to meet the labeling requirement, a new internal reflection IR procedure called negative second derivative is described and evaluated for the quantitation of total trans fat in the present study. The enhanced spectral features of a second derivative resolved issues that traditionally limited the sensitivity of the IR methodology. Calibration standard mixtures starting at approximately 0.5% trielaidin in the total fat (tripalmitin or triarachidin) were successfully generated and used to determine the trans fat levels for unknown test samples with trans content as low as approximately 1% of total fat. Quantitative IR data were compared to those obtained by gas chromatography and were found to be in good agreement.