Putative PFI1625c was cloned, over-expressed and purified to homogeneity. It is a 56.2 kDa monomeric protease which preferentially catalyzes the degradation of gelatin with a K = 30μM. It is a slow acting enzyme with optimal pH 8.5 and temperature 37 °C, and activity is sensitive to metalloprotease inhibitor 1,10-phenanthroline. PFI1625c active site was probed with a series of heterocyclic compounds and three molecules namely, BNPC-Inh2, DDBM-Inh1 and BHPM-Inh1 from the series were inhibiting PFI1625c protease activity. These heterocyclic compounds were found to irreversible inhibiting PFI1625c protease activity. Parasite culture was treated with these inhibitors and PFI1625c isolated from culture was found to be inactive without affecting other gelatinases present in the parasite. These inhibitors were used to generate chemically knockout PFI1625c in the parasite. PFI1625c knockout parasite remained at ring stage and was unable to complete its erythrocytic schizogony. Also, these knockout parasites were incapable to multiply. More careful analysis indicate these parasites develop oxidative stress as evident by the increase in lipid peroxidation, protein-carbonyl and a decrease of GSH level. In summary, the current study has employed biochemical, computational and pharmacological approaches to explore the role of PFI1625c in the parasite, its utility as a potential drug target to develop anti-malarials.