At present, the world faces severe issues of energy crises and environmental deterioration. Fuel is inevitable for industrial development and growth of any country. The fossil fuel resources have always been terrifying. Biofuel energy, a renewable source, seems to be an ideal solution for global energy and environmental concern. The present work focuses on an optimized protocol for the production of biodiesel from a feasible source, i.e., sesame plant oil (Sesamum indicum L.) through base catalyzed transesterification. The sesame plant oil is investigated in this study as a potential source of biodiesel based on fuel properties and physico-chemical analysis. The free fatty acid number of crude sesame oil was 0.83 mg/g of KOH (1.66%). Conversion of sesame crude oil (triglyceride) to biodiesel (fatty acid methyl ester) was 75.45% and 72.56% at 1:6 molar ratio (oil:methanol) by using optimum 0.67% KOH and 0.34% NaOH catalyst, respectively, at 60 degrees C for qualitative authentication. Specified analytical techniques are as follows: GC-MS used to clarify the different fatty acid methyl esters; FT-IR to monitor the transesterification reaction; and NMR for the justification of proton and carbon of that compound. Qualitatively, the biodiesel fuel properties were analyzed by the standard method American Society for Testing Material (ASTM).