Abstract: Biodiesel from four different renewable resources was produced. An optimization of the reference methodology by gas chromatography was conducted based on the construction of calibration curves for each biodiesel during fatty acid methyl esters (FAME) quantification. Therefore, in the proposed optimized methodology, pure commercial standards are not necessary. Consequently, a reduction in the research expenses is achieved, making this methodology a cost-effective alternative for FAME quantification. The calibration curves obtained for each biodiesel presented slope values between 0.5–0.7 and regression coefficients (R2) > 0.98 in all cases. These results were compared to those obtained by the conventional methodology. With respect to the validation of the optimized methodology, a comparison of the results obtained by this proposed methodology and the reference one is presented. Finally, a robust and promising technique to quantify FAME present in biodiesel was successfully developed in this study.