Background: Plasmodium falciparum and P. vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay (SMFA) to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. Methods: Transgenic murine malaria parasites (P. berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). Results: We describe the development of transmission competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. Conclusions: The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation.