Abstract Background Targeting the asymptomatic liver stage of Plasmodium infection through chemoprevention could become a key intervention to reduce malaria-associated incidence and mortality. Methods M5717, a Plasmodium elongation factor 2 inhibitor, was assessed in vitro and in vivo with readily accessible Plasmodium berghei parasites. In an animal refinement, reduction, replacement approach, the in vitro IC₉₉ value was used to feed a Population Pharmacokinetics modelling and simulation approach to determine meaningful effective doses for a subsequent Plasmodium sporozoite-induced volunteer infection study. Results Doses of 100 and 200 mg would provide exposures exceeding IC₉₉ in 96 and 100% of the simulated population, respectively. Conclusions This approach has the potential to accelerate the search for new anti-malarials, to reduce the number of healthy volunteers needed in a clinical study and decrease and refine the animal use in the preclinical phase. Graphical Abstract