Phenotypic drug screening has transformed the antimalarial development pipeline, facilitating the discovery of compounds targeting unexplored parasite proteins at a drastically accelerated rate. Breaking the cycle of malaria transmission is crucial to eradication and centres around inhibition of the sexual stage gametocytes and gametes. Transmissible sexual stages represent a population bottleneck of the Plasmodium life-cycle which face little selective pressure and are hence a desirable target of drug intervention. Recently discovered in a phenotypic screen, the N-((4- hydroxychroman-4-yl)methyl)-sulphonamide (N-4HCS) compounds potently inhibit P. falciparum male gamete formation (microgametogenesis) in the mosquito. By combining study of the remarkable Plasmodium gametocyte cellular biology with medicinal chemistry and chemical proteomics, here we performed a detailed analysis of N-4HCS compound activity. Adhering to a traditional drug developmental pipeline, we first performed N- 4HCS hit-to-lead development and yielded compounds with activity in the nanomolar concentration range and favourable safety profiles. Furthermore, an N-4HCS photoaffinity probe was utilised in a proteome-wide photoaffinity labelling study, identifying the P. falciparum sexual stage-specific protein, Pfs16, as the compound target. Notably, the Cellular Thermal Shift Assay confirmed label-free specificity of N-4HCS compounds to Pfs16. Flow cytometry and phase-contrast, widefield- fluorescence and electron microscopy revealed compounds exclusively inhibited microgametogenesis, with a cellular phenotype consistent with published reports on the targeted gene disruption of Pfs16. We therefore present the 16kDa parasitophorous vacuole membrane protein, Pfs16, as a desirable antimalarial target and the N-4HCS compounds as potent candidates for future development. Additionally, a live-cell fluorescence microscopy workflow was devised to enable visualisation of microgametogenesis from the onset of activation through to exflagellation. Crucially, our imaging approach was applicable to drug discovery and is accessible to the malaria research community, thereby facilitating future study of transmission-blocking drug candidates. This thesis hence addressed the urgent requirement for novel antimalarial interventions, developing and aiding future discovery of transmission-blocking drugs. Open Access