Art analytical method has been developed using Fourier transform infrared spectroscopy (FTIR) to determine biodiesel content in the reaction mixture to monitor the transesterification reaction. It is also shown that it can be used to determine biodiesel content in biodiesel-petrodiesel blends. The method with small modifications can also be used to determine the oil content in the adulteration of biodiesel-petrodiesel blends. Soybean oil is used as the model oil, and its methyl ester is used as biodiesel. The method uses a software viz. Enformatic FTIR Collection Manager (EFCM) to develop a calibration model and predict the concentration of biodiesel and oil. The software uses the nonlinear classical least square (CLS) method for calibration. It is shown that the method can be used to measure the amount of biodiesel accurately to the extent of 98.11% accuracy for biodiesel-oil mixtures and biodiesel content in the biodiesel-petrodiesel mixture (blend) with an accuracy of 99.99%. The method has also been used to determine the oil content and biodiesel content in the biodiesel-petrodiesel-oil mixture (blend adulteration) with an accuracy of about 95.32%.