Plasmodium vivax (Pv) is the most prevalent cause of malaria-associated morbidity outside of Africa. However, significant challenges in propagating the life cycle of Pv in vitro limit the scope of antigen and drug discovery, and availability of suitable models for interventional testing. Our goal is to develop a Pv vaccine using whole-sporozoite (SPZ) based approach similar to PfSPZ, that prevents all blood stage infections with Pv, and produce quality-controlled stocks of cryopreserved PvSPZ to promote well-controlled, reproducible studies in Pv including controlled human malaria infection (CHMI). To this end we have, 1) Demonstrated the capacity to repeatedly manufacture vialed, cryopreserved PvSPZ from mosquitoes fed on blood from Saimiri boliviensis (Sb), vialing as many as 80 million PvSPZ from 2000 mosquitoes in 1 day; 2) Developed assays to test and characterize PvSPZ for infectivity in vitro in traditional monolayer and in micro-patterned co-cultured primary human hepatocytes over 3-21 days; 3) Tested functional activity of drugs on inhibiting liver stage parasite development in vitro; 4) Infected NHPs in vivo; 5) Standardized methods to propagate Pv in Sb maintained as specific pathogen free (SPF); 6) Vialed aseptic, purified, infectious PvSPZ from aseptic mosquitoes fed on Pv-infected blood from SPF animals; and 7) Demonstrated that vialed, cryopreserved PvSPZ, Chesson strain infect humanized FRG KO huHep mice and produce hypnozoites in vivo. With an overall manufacturing process analogous to production of aseptic purified, cryopreserved PfSPZ, the PvSPZ products of 4 pilot runs conformed to all in-process, asepticity, in vitro potency, and release criteria, constituting a significant milestone towards regulatory compliance for human use. This enabling technology is intended to support development and testing of anti-Pv drugs and vaccines in CHMIs world-wide, and also forms the basis of a powerful vaccine approach to preventing Pv malaria when administered with anti-malarial chemoprophylaxis, the PvSPZ chemoprophylaxis vaccine (PvSPZ-CVac).