Understanding the mode of action of antimalarials is central to optimizing their use and the discovery of new therapeutics. Currently used antimalarials belong to a limited series of chemical structures and their mechanisms of action are coutinuously debated. Whereas the involvement of reactive species that in turn kill the parasites sensitive to oxidative stress, is accepted for artemisinins, little is known about the generation of such species in the case of quinolines or hydroxynaphtoquinone. Moreover, the nature of the reactive species involved has never been characterized in Plasmodium-infected erythrocytes. The aim of this work was to determine and elucidate the production of the primary radical, superoxide in Plasmodium-infected erythrocytes treated with artemisinin, dihydroartemisinin, chloroquine and atovaquone, as representatives of three major classes of antimalarials. The intracellular generation of superoxide was quantified by liquid chromatography coupled to mass spectrometry (LC-MS). We demonstrated that artemisinins, atovaquone and to a lesser extent chloroquine, generate significant levels of superoxide radicals in Plasmodium falciparum sensitive strains. More so, the production of superoxide was lowered in chloroquine-resistant strain of Plasmodium treated with chloroquine. These results consolidate the knowledge about the mechanism of action of these different antimalarials and should be taken into consideration in the design of future drugs to fight drug-resistant parasites.