Transesterification of triglycerides with short chain alcohols is the key reaction in biodiesel production, in addition to other applications in chemical synthesis. However, it is crucial to optimize reaction conditions to make enzymatic transesterification a cost-effective and competitive process. In this work, a new, easy Fourier transform infrared (FTIR) spectroscopic approach for monitoring the transesterification reaction is reported and compared with a gas-chromatographic method. The concentration of the total methyl esters in the reaction mixture is determined from the peak intensity at ∼1435 cm–1 in the second derivatives of the FTIR absorption spectra using a linear regression calibration. Interestingly, we found that the use of second derivatives allows an accurate determination of the methyl esters without the interference of free fatty acids. Moreover, information on substrate hydrolysis can be obtained within the same measurement by the infrared absorption at ∼1709 cm–1. We applied this approach to monitor methanolysis and hydrolysis reactions catalyzed by different commercial lipases, which displayed different sensitivities to methanol inhibition. Therefore, the FTIR approach reported in this work represents a rapid, inexpensive, and accurate method to monitor enzymatic transesterification, requiring very limited sample preparation and a simple statistical analysis of the spectroscopic data.