Shigella flexneri 2a is one of the most pathogenic bacteria among the Shigella spp., which is responsible for dysentery and causes masses of deaths throughout the world per year. A proper identification of the potential drug targets and inhibitors is crucial for the treatment of the shigellosis due to their emerging multidrug resistance (MDR) patterns. In this study, a systematic subtractive approach was implemented for the identification of novel therapeutic targets of S. flexneri 2a (301) through genome-wide metabolic pathway analysis of the essential genes and proteins. Ligand-based virtual screening and ADMET analyses were also made for the identification of potential inhibitors as well. Initially, we found 70 essential unique proteins as novel targets. After subsequent prioritization, finally we got six unique targets as the potential therapeutic targets and their three-dimensional models were built thereafter. Aspartate-β-semialdehyde dehydrogenase (ASD), was the most potent target among them and used for docking analysis through ligand-based virtual screening. The compound 3 (PubChem CID: 11319750) suited well as the best inhibitor of the ASD through ADMET and enzyme inhibition capacity analysis. To end, we hope that our proposed therapeutic targets and its inhibitors might give some breakthrough to treat shigellosis efficiently in in vitro.