This work aims at determining the acid number of biodiesel from three different matrices (soy/animal fat, castor oil, and palm oil) and using three different analytical methods based on automatic volumetric titration with potentiometric detection. One of the methods is described in the ASTM D664 Standard, and the other two were similar to the former, but changing the solvent (2-propanol by a mixture of toluene, 2-propanol, and water with a volumetric ratio of 100:99:1) or the filling solution (LiCl by KCl) of the combined glass electrode used in the detection. This study was carried out in order to assess the influence of the combined glass electrodes and solvents in determining the acid number of biodiesel. The analyses were carried out in one laboratory, so it has an exploratory nature. The ordinary statistical tests were applied to the data (F-test and t-test) in order to compare the results from the three methods. Also, an extensive statistical analysis was not performed. Basically, it was observed that there was a difference of about 3.1 % between the acid number obtained by the three methods. In addition, the method described in the ASTM D664 Standard showed a repeatability of 4.4 % for soy/animal fat biodiesel, 3.9 % for castor biodiesel, and 9.8 % for palm biodiesel, which are comparable to literature data.