Glycerol is a typical byproduct of the Colombian biodiesel industry, which is usually released within the process of effluents. However, glycerol could be a valuable substrate for anaerobic degradation and biogas production. This study addresses the biochemical potential of methane (BPM) in glycerol solutions produced in transesterification facilities in Colombia, by using sludge from the effluent treatment of palm oil mills as inocula. The BPM technique was developed with in vitro tests (batch mode) based on an experimental design 32, with three-factor levels, being the minimum (−1), the central (0), and the highest (1) for each factor, and two design factors (substrate concentration and substrate/inoculum ratio). BPM tests were carried out in a volume of 300 mL for 10 days. Glycerol solutions were prepared with deionized water and their concentration was expressed as chemical oxygen demand (COD) per liter (mg COD/L). The substrate/inoculum ratios were established between 2.0 and 4.0, and two sets of experiments were carried out at distinct temperatures of 23 °C and 37 °C. The biogas generation volume was measured by the simple displacement method. The influence of pH depends on the pretreatment of the inoculum by heating it at 110 °C for 15 min, allowing a decrease of 2 pH units starting from 6.5 units on day zero and for the second day of evaluation (4.6 at 37 °C and 5.1 at 23 °C), stabilizing the reaction in an acidic medium toward the production of CO2 and not CH4 during the time measurement. Without the inoculum treatment, the pH decreased by 1.5 units during the evaluated days. Likewise, it was determined that with the pretreatment of the inoculum, it was possible to obtain a higher biogas volume of 88% at 23 °C and 80% at 37 °C compared to the BPM without heating the inoculum. Finally, a higher COD removal obtained was 56% at 23 °C and 59% at 37 °C without preheating the inoculum.