Different pretreatment methods (milling, ultrasonication, liquid hot water, and alkali hydrolysis) were evaluated on rice straw and coconut shell biomass as a substrate for biogas production for vehicle fuel, provide heat and generate electricity. This was achieved by the sequential combination of ultrasonication, hot water, and alkali hydrolysis treatment at (4% and 5% w/v) concentration was evaluated. Additionally, Fourier Transform Infrared (FT-IR) spectroscopy was used in identifying structural differences and identification of compounds in the treated biomass. The results show that the combination of ultrasonication-alkali hydrolysis, hot liquid water using 3% of NaOH improved methane yield of 150% for rice straw and 290% for coconut shell compared with the original treatment. Moreover, the Fourier-transform infrared spectroscopy (FT-IR) methodology identified modifications in the biomass structure after different types of pretreatment and conditions. The results of this research are the initial steps for the development of new processes using the Ultrasonication-NaOH LHW pretreatment, for the production of biogas from agricultural biomass waste.