A large research effort is currently underway to find an effective and affordable malaria vaccine. Tools that enable the rapid evaluation of protective immune responses are essential to vaccine development as they can provide selection criteria to rank order vaccine candidates. In this study we have revisited the Inhibition of Sporozoite Invasion (ISI) assay to assess the ability of antibodies to inhibit sporozoite infection of hepatocytes. By using GFP expressing sporozoites of the rodent parasite P. berghei we are able to robustly quantify parasite infection of hepatocyte cell lines by flow cytometry. In conjunction with recently produced transgenic P. berghei parasites that express P. falciparum sporozoite antigens, we have been able to use this assay to measure antibody mediated inhibition of sporozoite invasion against one of the lead malaria antigens P. falciparum CSP. By combining chimeric rodent parasites expressing P. falciparum antigens and a flow cytometric readout of infection, we are able to robustly assess vaccine-induced antibodies, from mice, rhesus macaques and human clinical trials, for their functional ability to block sporozoite invasion of hepatocytes.