BACKGROUND: The infectivity of Plasmodium gametocytes is typically determined by microscopically examining the midguts of mosquitoes that have taken a blood meal containing potentially infectious parasites. Such assessments are required for the development and evaluation of transmission-reducing interventions (TRI), but are limited by subjectivity, technical complexity and throughput. The detection of circumsporozoite protein (CSP) by enzyme-linked immunosorbent assay (ELISA) and enhanced chemiluminescent slot-blot (ECL-SB) may be used as objective, scalable alternatives to microscopy for the determination of infection prevalence. METHODS: To compare the performance of the CSP ELISA and ECL-SB for the detection of mosquito infection, four groups of Anopheles stephensi mosquitoes were infected with cultured Plasmodium falciparum gametocytes. At day-8 post-infection (PI), parasite status was determined by microscopy for a sample of mosquitoes from each group. At days 8 and 10 PI, the parasite status of separate mosquito samples was analysed by both CSP ELISA and ECL-SB. RESULTS: When mosquito samples were analysed 8 days PI, the ECL-SB determined similar infection prevalence to microscopy; CSP ELISA lacked the sensitivity to detect CSP in all infected mosquitoes at this early time point. When mosquitoes were analysed 48 h later (10 days PI) both assays performed as well as microscopy for infection detection. CONCLUSIONS: Whilst microscopical examination of mosquito guts is of great value when quantification of parasite burden is required, ECL-SB and CSP ELISA are suitable alternatives at day 10 PI when infection prevalence is the desired endpoint, although CSP ELISA is not suitable at day 8 PI. These results are important to groups considering large-scale implementation of TRI.