Plasmodium falciparum (P. falciparum) infections leading to malaria have severe clinical manifestations and high mortality rates with approximately 200 million cases in 2015. Chloroquine (CQ), a former crucial antimalarial chemotherapy, has been deemed ineffective due to wide-spread resistance which compromises its efficacy in parasite hemozoin inhibition. Recent studies, however, have unveiled a novel mode of action in which low micromolar levels of CQ permeabilized the parasite’s digestive vacuole (DV) membrane, leading to calcium efflux, mitochondrial depolarization and parasite DNA degradation. These phenotypes resemble that of classical mammalian apoptosis, thereby alluding to CQ-mediated DV rupture as a potential parasiticidal pathway for exploitation through the screening for and discovery of DV-disruptive antimalarials. Against this backdrop, a target-based phenotypic screening on the Malaria Venture (MMV) ‘Pathogen Box’ compound library was performed for the identification of potent DV-disruptive compounds relative to CQ. The hits obtained were further characterized on their dose-response potencies across multiple parasite strains and rate of parasiticidal activity to select for rapid-acting, non-cross resistant and highly potent top hits from the ‘Pathogen Box’ repository. Consequently, MMV085071 emerged as the most promising compound. Subsequent hit-to-lead generation through SAR analysis and assessment of in vitro efficacies via downstream MMV085071 analogues synthesis led to the identification of analogue MMV1545561 with over 5-fold increase in in vitro parasiticidal activity. From the drug development perspective, the aims and findings of the project could offer an alternative first-in-class chemotherapy with novel molecular entities and scaffolds in view of antimalarial resistance, at the same time while aiming to enhance the understanding of P. falciparum pathophysiology.