One of the major mechanisms followed by the therapeutic agents to target the causative organism of TB, Mycobacterium tuberculosis, involves disruption of its DNA replication cycle. The process of replication involves two steps, i.e., breakage and reunion of DNA at gyrase A (GyrA) domain and ATP hydrolysis at gyrase B (GyrB) domain, both occur simultaneously. Current therapy for multi-drug resistant TB (MDR-TB) involves FDA approved, fluoroquinolone-based antibiotics, which act by targeting replication process at GyrA domain. However, resistance against fluoroquinolones due to mutations in the GyrA domain has limited the use of this therapy and shifted the focus of research community on GyrB domain. Thus, in the present study novel chemotherapeutic agents for resistant TB were designed by exploring GyrB domain using in silico techniques. Pharmacophore model of GyrB domain was employed for screening an In-house database. Followed by cross-screening via a qualitative Hip-Hop pharmacophore model for GyrA to remove non-selective gyrase B inhibitors. Further molecular dynamics simulations and MM-GBSA calculations were performed to determine stability and binding affinity of the screened molecules. These analyses resulted in five putative oxindole based selective GyrB domain inhibitors, which were synthesized and evaluated for anti-tubercular activity against M. tuberculosis H₃₇Rv strain. Two compounds exhibited significant anti-TB activity, whereas other three compounds were found to be outliers of the in silico study.