The extraction and determination of alkaloids was performed and confirmed by phytochemical analysis. Six different quinazoline alkaloids (vasicoline, vasicolinone, vasicinone, vasicine, adhatodine and anisotine) were found in the leaf of Justicia adhatoda (J. adhatoda). The presence of the peaks obtained through HPLC indicated the diverse nature of alkaloid present in the leaf. The enzyme β-ketoacyl-acyl-carrier protein synthase III that catalyses the initial step of fatty acid biosynthesis (FabH) via a type II fatty acid synthase has unique structural features and universal occurrence in Mycobacterium tuberculosis (M. tuberculosis). Thus, it was considered as a target for designing of anti-tuberculosis compounds. Docking simulations were conducted on the above alkaloids derived from J. adhatoda. The combination of docking/scoring provided interesting insights into the binding of different inhibitors and their activity. These results will be useful for designing inhibitors for M. tuberculosis and also will be a good starting point for natural plant-based pharmaceutical chemistry.