AIM: To develop an in vitro assay incorporated with metabolism for screening antimalarials. METHODS: Antimalarial activities of chloroquine, pyronaridine, cycloproguanil and proguanil were tested with an in vitro assay based on the routine in vitro culture of P. falciparum incorporated with a metabolic system comprising rat liver microsomes and NADP cofactors. RESULTS: Except proguanil, the three drugs perse all showed antimalarial activities, but proguanil must be metabolized into its active metabolite for the antimalarial effect. CONCLUSION: The in vitro assay incorporated with metabolism could overcome the shortcoming of miss-screening the potential antimalarials which must be metabolically biotransformed into the active metabolite.