ABSTRACTIn present investigation, lipid extraction was performed from non-edible seeds of Indigofera colutea using Soxhlet technique with ultrasonic pre-treatment to evaluate maximum yield of oil. Different solvents were used for the extraction of oil from the biomass. Different parameters such as solvent, particle size, temperature, time, and solvent-to-solid ratio have been examined. The high yield of oil extraction was obtained at 6% moisture content, 0.15 mm particle size, 65°C temperature, 210 min, and 6:1 solvent-to-solid ratio using n-hexane as solvent. The maximum yield of oil (38.5%) was obtained from 75 g of I. colutea biomass. From Fourier transform infrared spectroscopy analysis, the methyl group–CH3 bond indicates the presence of asymmetric stretch. Fatty acid profile of the optimized I. colutea oil was analyzed using gas chromatography mass spectroscopy analysis. The unsaturated fatty acid content was found to be very high at 52.214%. I. colutea biomass proved to be a suitable source for the biodiesel production.