An alternative method for the production of biodiesel from processed fats derived from Category 1, 2 and 3 animal by-products was assessed. The method is based on a pre-cleaning process, acidic esterification/transesterification of tallow using 1.5% methanesulfonic acid w/w; 140°C; 5.5 bar absolute pressure (bara); 4 h, followed by fractional distillation. The application focuses on the capacity of the alternative method to inactivate prions. Given the limitations that biodiesel presents for direct measurement of prion infectivity, the BIOHAZ Panel considered, based on the outcome of previous EFSA Opinions and current expert evaluation, that a reduction of 6 log10 in detectable PrPSc signal would be necessary to consider the process at least equivalent to previously approved methods for Category 1 animal by-products. This is in addition to the inactivation achieved by the pressure sterilisation method applied before the application of any biodiesel production method. Experimental data were provided via ad hoc studies commissioned to quantify the reduction in detectable PrPSc in material spiked with scrapie hamster strain 263K, as measured by western blot, for the first two steps, with distillation assumed to provide at least an additional 3 log10 reduction, based on published data. Despite the intrinsic methodological caveats of the detection of PrPSc in laboratory studies, the BIOHAZ Panel considers that the alternative method, including the final fractional distillation, is capable of achieving the required 6 log10 reduction of the strain 263K PrPSc signal. Therefore, the method under assessment can be considered at least equivalent to the processing methods previously approved for the production of biodiesel from all categories of animal by-product raw materials. It is recommended to check the feasibility of the proposed HACCP plan by recording the main processing parameters for a certain time period under real industrial conditions.