The demand for renewable energy has increased significantly over the last few decades due to limited fossil fuel reserves and concern over their contribution to greenhouse gas emissions. To meet the demand for renewable energy and finding an alternative fuel source to fossil fuels, emphasis has been placed on the use of biofuels from biomass resources. The production of one of the biofuels, biodiesel results in the generation of crude glycerol at rate of 10% on weight basis. In order to maintain environmentally sustainable and economically viable biodiesel production, it is essential to find alternative use for crude glycerol. The goal of this research was to improve methane (CH₄) production from glycerol using coupled ozonation-anaerobic digestion process. To achieve the goal, the following objectives were completed: (1) investigated the enhancements in CH₄ production due to pre-ozonation of glycerol, (2) evaluated the effects of operational parameters, such as substrate to inoculum ratio and temperature, and (3) compared energy input vs. energy output of coupled ozonation-anaerobic digestion process. Anaerobic digestion experiments were conducted at bench-scale to test the feasibility of coupled ozonation-anaerobic digestion of glycerol. A glycerol solution ozonated for 5, 10, 15, and 20 minutes was subsequently anaerobically digested for 20 days. Results show an improvement in CH₄ production in this experiment compared to anaerobic digestion of non-ozonated glycerol. Anaerobic digesters containing glycerol ozonated for 20 minutes produced about 25% more CH₄ than that of the anaerobic digesters using non-ozonated glycerol. In this same set of experiments, the mL of CH₄/mg of COD oxidized varied in the range of 0.51 – 0.79. To examine the effect of the substrate to inoculum ratio on CH₄ production, anaerobic digestion experiments were conducted at substrate/inoculum ratios of 1:1, 1:1.5, and 1:2.3 using glycerol solution ozonated for 20 minutes as a substrate. The results showed that decreasing the substrate/inoculum ratio from 1:1 to 1:2.3 reduced the CH₄ production by 37.9%. Under the experimental conditions used in this study, glycerol ozonated for 5 min resulted in 7 kWh energy gain per kWh energy input, while 20 min of ozonation yielded 2 kWh of energy per kWh energy input. The implications of the findings in this research are that the proposed process could be a viable solution for the surplus crude glycerol generated during biofuels production. This process increases CH₄production, as well as reduce the amount of glycerol sent to landfills. Thus, the couple ozonation-anaerobic digestion process has the potential to contribute to the long-term economic viability and sustainability of biofuels.