Most current antimalarials for treatment of clinical Plasmodium falciparum malaria fall into two broad drug families and target the food vacuole of the trophozoite stage. No antimalarials have been shown to target the brief extracellular merozoite form of blood stage malaria. We studied a panel of 12 drugs, 10 of which have been used extensively clinically, for their invasion, schizont-rupture and growth-inhibitory activity using high throughput flow cytometry and new approaches for the study of merozoite invasion and early intra-erythrocytic development. Not surprisingly, given reported mechanisms of action, none of the drugs inhibited merozoite invasion in vitro. Pretreatment of erythrocytes with drugs suggested that halofantrine, lumefantrine, piperaquine, amodiaquine and mefloquine diffuse into and remain within the erythrocyte and inhibit downstream growth of parasites. Studying the inhibitory activity of the drugs on intra-erythrocytic development, schizont rupture and re-invasion enabled several different inhibitory phenotypes to be defined. All drugs inhibited parasite replication when added at ring-stages, but only artesunate, artemisinin, cycloheximide and trichostatin A appeared to have substantial activity against ring-stages, whereas the other drugs acted later during intra-erythrocytic development. When drugs were added to late schizonts, only artemisinin, cycloheximide and trichostatin A were able to inhibit rupture and subsequent replication. Flow cytometry proved valuable for in vitro assays of antimalarial activity, with the free merozoite population acting as a clear marker for parasite growth inhibition. These studies have important implications for further understanding the mechanisms of action of antimalarials, studying and evaluating drug resistance, and the development of new antimalarials.