<p>This review comprehensively summarizes the current state of in vivo antimalarial activity research. To address the resistance challenges that have emerged with almost all drugs, there is an immediate necessity for innovative drug options. In antimalarial drug discovery, in vivo, studies are essential for assessing the effectiveness, pharmacokinetics, and toxicity of prospective drugs under physiological circumstances. In contrast to in vitro studies, in vivo antimalarial assays replicate the physiological circumstances of the host, enabling researchers to investigate the interaction between the parasite and the host immune response, so offering a more thorough evaluation. This review article offers a detailed and systematic components of various well-established in vivo models, providing critical insights into their methodologies, applications, and relevance in advancing antimalarial research, as well as a comprehensive analysis of the folate biosynthesis pathway elucidating its mechanistic role as a critical target for antimalarial drug development. Moreover, it offers a thorough review of currently approved antimalarial agents within the Medicines for Malaria Venture (MMV) portfolio, highlighting their clinical efficacy and relevance in overcoming drug resistance.</p> Graphical Abstract <p></p>