Malaria is a disease of significant and ongoing global burden, caused by parasites of the Plasmodium genus and carried by Anopheles mosquitoes. Plasmodium falciparum is responsible for 90% of global malaria mortality. Effective drug treatment is instrumental to control of this disease, and the World Health Organisation (WHO) recommends artemisinin (ART) combinations for treatment of uncomplicated P. falciparum malaria. In this process of treatment, intentionally or otherwise, ART may be coadministered with antibiotics such as doxycycline or clindamycin. Previous work in the Ralph laboratory assessed that these antibiotics have an antagonistic effect on ART. The key to this antagonism lies in a relict plastid organelle in Plasmodium parasites known as an apicoplast. This endosymbiotic organelle is the target of a number of antibiotic therapies due to its bacterial-type translation machinery. These antibiotics disrupt apicoplast function, reducing isoprenoid synthesis, and ultimately result in the parasites’ inability to traffic haemoglobin into their digestive vacuole, where its breakdown would otherwise release free haem. Crucially, the iron in this free haem is necessary in activating artemisinin derivatives to produce free radicals leading to parasite death. In combining these drug classes, we posit that artemisinin activation will be impaired, contributing to the observed antagonist effect between apicoplast-targeting antibiotics and ART derivatives. To test this, we are performing a novel, mass spectrometry-based approach to directly measure artemisinin activation within Plasmodium trophozoite-stage parasites pre-treated with apicoplast-targeting antibiotics. By way of this assay, this project aims to shed light on the nature of antagonism between these antibiotics and DHA and may inform how the drugs should be used in the future.