Preventing malaria parasite transmission to the mosquito vector is a key elimination challenge that could benefit from a diagnostic test that can identify transmission-competent individuals. Only sexual parasite forms, called gametocytes, which activate into gametes (gametogenesis) upon mosquito uptake or blood sampling are responsible for transmission. Herein, we devised a nanozyme-based immunoassay to detect the Plasmodium falciparum female gametocyte activation antigen PfG377, which is released during gametogenesis. Initial validation of our nanozyme assay with cultured parasites demonstrated that levels of PfG377 were higher in supernatants of activated versus nonactivated cultures and those treated with transmission blocking drugs. To define the field potential of this approach, patient samples from a clinical transmission-focused trial were used, including those receiving treatment with the gametocytocidal drug primaquine (PQ) that sterilizes gametocytes and blocks transmission. PQ-treated patient samples showed significantly lower signals of PfG377 2 days after treatment, consistent with an inability of PQ-treated gametocytes to activate and release antigen upon blood sampling. This study serves as a pathfinder for field transmission rapid diagnostics to detect transmission-competent individuals, which could help revive malaria elimination strategies.