Rift Valley fever virus (RVFV), a recognized category A priority pathogen, causes large outbreaks of Rift Valley fever with some fatalities in humans in humans and huge economic losses in livestock. As wild-type RVFV must be handled in BSL-3 or BSL-4 laboratories, we constructed a high-titer vesicular stomatitis virus (VSV) pseudotype bearing RVFV envelope glycoproteins to detect neutralizing antibodies in vitro under BSL-2 conditions. The neutralizing properties of 39 amino acid mutant sites that have occurred naturally over time in the RVFV envelope glycoproteins were analyzed with their corresponding pseudoviral mutants separately. Compared with the results in the primary strain, the variants showed no statistically significant differences. We next established a Balb/c mouse pseudovirus infection model for detecting neutralizing antibodies against pseudovirus. Five immunizations with pseudoviral DNA protected the mice from infection with the pseudovirus. Bioluminescence imaging, which we used to evaluate viral dissemination and distribution in the mice, showed a good relationship between the neutralizing antibodies titers in vitro. These pseudovirus methods will allow for the safe determination of neutralizing antibodies in vivo and in vitro, and will assist with studies on vaccines and drugs against RVFV with the long term objective of Rift Valley fever prevention.