Mycobacterium tuberculosis, the causative agent of tuberculosis, has been a serious threat to public health. The recent emergence of extreme drug-resistant strains further worsened the situation and required more serious efforts to find a better cure for TB. Availability of the complete genome map of various Mycobacterium tuberculosis strains serves a basis to propose new therapeutic targets against Mycobacterium tuberculosis. The present study aimed to identify and characterize potential and novel drug target against Mycobacterium tuberculosis by using an in silico approach. The study consisted of three phases. In phase I, subtractive/comparative genomics was performed on genomic data of 17 strains of Mycobacterium tuberculosis comprised of 66,988 protein sequences. The analysis resulted in three identical hypothetical proteins of distinct origin. The shortlisted sequences were non-homologous to the host, a prerequisite characteristic of potential drug target. One of the sequences, i.e., WP_003901628.1 (the locus tag name is CFBR_RS17985) from the CDC5180 strain was further subjected to other bioinformatics methods in phase II. In phase II, the shortlisted protein sequence was qualitatively characterized for subcellular localization, functional family prediction and evolutionary relationships. The predicted subcellular localization of the query protein was found in cytoplasmic region. The functional family classification resulted in classifying the protein in transferase superfamily. Finally, in phase III of the study, the structure-based methods (i.e., homology modeling and docking) were performed to build the three-dimensional (3D) structure and to predict the putative binding site. The PDB-ID 3DR4 was found to be a suitable template having 30.1% sequence identity. The binding site of modeled protein was deciphered with DoGSite scorer tool. Later on, the ProBis tool was used to probe structurally similar binding sites in Protein Data Bank. Subsequently, the co-crystal ligands of the Protein Data Bank structures (prioritized by high-rank Z scores) were extracted and cross-docked into the binding site of the modeled protein. The cross-docking results revealed two ligands, i.e., 4'-deoxy-4'-aminopyridoxal-5'-phosphate and OG1 from two Protein Data Bank structures, i.e., 1MDO and 4K2M as best docked with RMSDs 2.8 and 2.5 , respectively. It is inferred from the protein structure that the identified protein is alanine-rich with a PI < 7. Current study convincingly proposed that the shortlisted hypothetical protein (WP_003901628.1) is a potential drug target against which new drug candidates could be designed in a hope to cure tuberculosis.