A series of N- (4-Substituted Benzylidine)-amino-5-pyridine-3-yl-1,3,4-triazol-2-thiol (3a-f) were synthesized from Isoniazid. In literature survey revealed that, isoniazid was found to interfere with Nicotinamide adenine dinucleotide (NAD)-utilizing enzymes, primarily the enoyl-ACP reductase encoded by the InhA gene, leading to the arrest of mycolic acid synthesis, which is essential to M. tuberculosis. InhA enzyme was chosen based upon its hydrophobic properties that favorably interact with thioamide or thiourea moieties. All the synthesized compounds are having sulphahydryl group which shows better interaction with active site of M. Tuberculosisenoyl reductase enzyme encoded with the gene InhA. Synthesized compounds were characterized and evaluated for in-vitro anti-TB activity against M.TbH37Rv strain by Alamar-Blue assay. The results expressed as MIC (minimum inhibitory concentration) in mg/ml. Antitubercular assay has been carried out at Micropharm Diagnosis Center, Gandhinagar. Among the six compounds 3f has shown highest docking and gliding score (-7.172, -7.306) and highest activity (MIC 6.25 mg/ml).