Malaria remains one of the deadliest human infectious diseases. In 2020 alone, there were an estimated 241 million clinical cases of malaria which resulted in approximately 627,000 deaths. With 3.2 billion people at risk of malaria and a global agenda focused on eradication there is a desperate need to improve malaria prevention and control strategies. As there is no highly effective vaccine, the treatment and prophylaxis of malaria are dependent on chemotherapy which is associated with increasing levels of parasite resistance. Given the lengthy development times for new drugs, strategies to safeguard current drugs are essential. This includes the development of resistance monitoring strategies and the rational selection of combination partners which should ideally be guided by mode of action information. The antimalarial drug proguanil is used in combination with the cytochrome bc1 inhibitor atovaquone (as Malarone®) for malaria prevention and treatment. While proguanil was developed as a pro-drug and is metabolized in vivo to the potent dihydrofolate reductase (DHFR) inhibitor cycloguanil, recent studies in our laboratory have demonstrated that it, and a cyclization-blocked analogue (tBuPG) that cannot be metabolized to cycloguanil, have potent slow action antiplasmodial activity independent of cycloguanil. Data has demonstrated that this activity is different to other slow action antimalarial agents and preliminary unpublished metabolomics work has shown that deoxythymidine monophosphate (dTMP) accumulates in proguanil and tBuPG treated parasites raising the hypothesis that P. falciparum thymidylate kinase (PfTMPK), the enzyme responsible for conversion of dTMP to deoxythymidine diphosphate (dTDP) may be a target of proguanil. However, the mechanism of proguanil’s slow action activity remains unknown. As proguanil’s intrinsic slow action activity may have clinical implications and there have been recent reports of resistant P. falciparum parasites in Uganda, the current work aimed to further investigate the slow action activity of proguanil. Investigations included examining the role of P. falciparum thymidylate kinase (PfTMPK) in the mode of action of proguanil, investigating the genome of P. falciparum parasites selected for in vitro resistance to tBuPG and examining the thermostability of proteins in the presence and/or absence of proguanil. P. falciparum thymidylate kinase (PfTMPK) was investigated as a target of proguanil as preliminary metabolomics work performed prior to this project (unpublished; collaboration with Professor Malcolm McConville, Melbourne University) raised the hypothesis that this enzyme is a target of proguanil. However, when tested during the current study, supportive data for this hypothesis was not generated. The overexpression of PfTMPK in P. falciparum 3D7 parasites did not impact proguanil or tBuPG activity (proguanil IC50 values of 0.094 μM versus 0.145 μM; P>0.05; tBuPG IC50 0.058 μM versus 0.046 μM, P>0.05). Proguanil and tBuPG failed to stabilize PfTMPK using two separate CETSA approaches and proguanil was shown to have no direct inhibitory activity against PfTMPK in in vitro activity studies. While these studies were not without limitations, together they suggest that proguanil does not directly target PfTMPK. However, there is still the possibility that PfTMPK could be indirectly associated with proguanil action including impacting PfTMPK localization which could lead to reduced conversion of dTMP to dTDP. Alternatively, adenosine triphosphate (ATP), which is required for the conversion of dTMP to dTDP by PfTMPK, could be affected by proguanil. This would align with a previously proposed hypothesis that proguanil may target ATP synthase. However, an impact on ATP would be expected to result in pleiotropic metabolic effects, which were not seen. In addition, while metabolomics data provided information on total ATP pools and not specific sub-cellular changes (e.g., mitochondria), they remained unchanged in proguanil or tBuPG treated versus wildtype parasites.