In tropical and subtropical regions, in which it causes intense morbidity and mortality, malaria continues to pose an extreme threat to global health. The parasite Plasmodium, of which Plasmodium falciparum is the maximum virulent species that causes life-threatening infections, is disseminated with the aid of Anopheles mosquitoes. New antimalarial drugs are desperately required, as evidenced by the rise of chemoresistant Plasmodium variants, especially the ones which can be immune to artemisinin and chloroquine. Given their wide range of bioactive metabolites, including terpenoids, polysaccharides, and phenolic compounds, medicinal mushrooms are tested as a capacity source of antimalarial compounds. We offer a thorough method for isolating, purifying, and evaluating polysaccharides from mushrooms for their antimalarial properties using the SYBR green I fluorescence assay. This method permits fast and sensitive quantification of Plasmodium, making the assay ideal for high-throughput screening and antimalarial drug testing.