Targeting the crucial step of Plasmodium transition from vertebrate host to mosquito vector is a promising approach to eliminate malaria. Uptake by the mosquito activates gametocytes within seconds, and in the case of male (micro) gametocytes leads to rapid DNA replication and the release of eight flagellated gametes. We developed a sensitive assay to monitor P. berghei microgametocyte activation based on [3H]hypoxanthine incorporation into DNA. Optimal pH range and xanthurenic acid concentrations for gametocyte activation were established and the kinetics of DNA replication investigated. Significance of the method was confirmed using P. berghei mutants and the assay was applied to analyse the effect of protease inhibitors, which revealed differences regarding their inhibitory action. The developed method thus appears suitable for reproducible determination of microgametocyte activation, medium-throughput drug screenings and deeper investigation of early blocks in gametogenesis and will facilitate the analysis of compounds for transmission blocking activities.