The current study was aimed to identify new inhibitors against tryptophan synthase from Mycobacterium tuberculosis for anti-tuberculosis drug discovery. The α-subunit of tryptophan synthase being unique and unexplored protein target was selected for discovery of new inhibitors that can serve as leads for anti-TB drugs discovery with novel mode of action. Here a combination of computational and experimental approaches was utilized. Both the structure based (SB) and ligand based (LB) virtual screening approaches were employed for prediction and identification of new hits as inhibitors against α-subunit of tryptophan synthase from M. tuberculosis. The structure based virtual screening (SBVS) of eMolecules database was done against homology model for protein i.e., α-subunit of tryptophan synthase. Seven new inhibitors were identified on the basis of their binding score, binding interactions and physiochemical properties. Similarly, ligand based virtual screening (LBVS) was used for screening of “clean drug like” subset of ZINC database against pharmacophore model. This pharmacophore model was generated using the structures of already reported inhibitors of the α-subunit of TrpS from literature and hits were identified in terms of rmsd (<1). Later the molecular docking studies of hits into the active pocket resulted in identification of five new inhibitors against α-subunit of TrpS. The anti-tuberculosis activity of proposed inhibitors identified through SBVS and LBVS was performed using agar dilution method and LJ media based method that resulted in identification of a new benzamide inhibitor and ZINC09150898. The benzamide inhibitor showed 100% growth inhibition of H37Rv strain of M. tuberculosis at 25 μg/mL and considerable anti-mycobacterial effect up to 6 μg/mL in whole cell based activity. The second new inhibitor i.e., ZINC09150898 showed antibacterial activity against H37Rv strain of M. tuberculosis at the concentration of 50 µg/mL (100% inhibition) and partial inhibition up to 12 µg/mL. The binding stability of both benzamide inhibitor and ZINC09150898 inside the binding pocket was further investigated through MD simulation studies involving RMSD, RMSF and secondary structure analysis that showed no major structural fluctuations in protein structure during the explored time scale. The current study can be further extended for enzyme based assay evaluation and could be considered as candidate for drug discovery against tuberculosis. In our attempt to find out new lead candidates for anti-tuberculosis drug discovery we performed in silico comparison of putative drug binding pockets of twelve essential metabolic enzymes from M. tuberculosis and other bacterial pathogens belonging to the ESKAPE group. The aim of this comparative analysis was to provide guidelines for the likelihood of transferability of the inhibitors from one species to another. In this comparison pathways other than tryptophan biosynthesis were investigated and the selection of targets was based on availability of the 3D-structure. Drug binding pockets of these essential enzymes from selected metabolic pathways that were conserved across ESKAPE pathogens, M. tuberculosis, M. smegmatis and E. coli were compared using their reported 3D structures and amino acid sequence alignment. This comparative analysis showed that drug binding pockets of these enzyme are mostly identical with good sequence identity (70-100 %) across different species used in this study suggesting that inhibitor designed for enzyme target of one species may have chances to inhibit same enzyme target in other species as well. This study suggested that antibiotics targeting enzyme of one pathogen might have similar inhibitory potential against other bacterial species as well if their binding pockets are conserved and can effectively help in design of new antibiotics against M. tuberculosis and ESKAPE pathogens as well as.