Plasmodium falciparum is the causative agent of the most severe form of human malaria. The rise in parasite resistance to existing antimalarials poses an immediate need for drugs with novel biological targets. To expand our repertoire of antimalarial drug targets we focused on one member of the aminoacyl-tRNA synthetases, the cysteinyl-tRNA synthetase (PfCysRS). We demonstrated that the single PfCysRS transcript is alternatively spliced and show that PfCysRS isoforms traffic to the cytoplasm and apicoplast using a combination of endogenous and heterologous tagging experiments in both P. falciparum and Toxoplasma gondii. PfCysRS is able to recognise and charge the eukaryotic tRNACys encoded by the Plasmodium nucleus as well as the bacterial type tRNA encoded by the apicoplast genome, albeit with a preference for the eukaryotic type cytosolic tRNA. Therefore, inhibitors of the Plasmodium dual-targeted CysRS would potentially offer a new therapy capable of the desirable immediate effects to parasite growth as well as the irreversibility of inhibitors that disrupt apicoplast inheritance. We performed in silico docking to identify putative PfCysRS inhibitors that inhibit growth of P. falciparum parasites in culture.bitors for drug development.Compounds displaying low micromolar (<10 μM) inhibition by parasite growth assays were further tested using genetic and biochemical approaches to validate these compounds as true cysteinyl-tRNA synthetase inhibitor for drug development.