One of the main diseases caused by Mycobacterium tuberculosis is tuberculosis (TB). It is necessary to create safer, smaller chain peptides in order to prevent TB from becoming widely and multi-drug resistant. Two fatty acid synthase systems (FAS), FAS-I and FAS-II, are involved in the production of mycolic acids, which are long-chain, acylated, and hydroxylated fatty acids (MA). Our investigation focuses on mycolic acid synthase, which is essential for Mycobacterium tuberculosis intracellular survival and lowers the apoptotic activity of macrophages. The primary lipid in the envelope, mycolic acid, makes up the exterior mycomembrane. Our goal is to investigate proline-based tripeptides as effective mycolic acid synthase inhibitors. As a crucial amino acid, proline has been used in various shorter chains of selected tripeptides for a thorough analysis of 3D interactions using the Swiss DOCK online tool (PDB code: 1l1e), Chimera, and Discovery studio visualizer. With a G value of (-8.92 kcal/mol), Pro-trp-tyr was determined to be the most promising lead as a powerful mycolic acid synthase inhibitor, followed by Pro-glu-gln and Pro-gly-his, which had G values of (-8.66 kcal/mol) and (-8.62 kcal/mol), respectively.