We have developed retroviral vectors that can be used in neutralisation assays to determine antibody titres for biohazard level 3/4 pathogens. This assay has two advantages over existing methods: (1) Retroviral pseudotypes can be handled in biohazard level 2 laboratories; (2) The use of either green fluorescent protein, luciferase or β-galactosidase as a reporter allows the assay to be used at low cost in laboratories throughout the world. G-protein sequences from CVS-11, EBLV-1, EBLV-2 and a human street genotype-1 virus (RV61) cDNA were cloned and co-expressed along with HIV/MLV gag-pol and a GFP or luciferase in human 293T cells. The resulting pseudotyped viruses were able to infect a number of target cells and, with the exception of RV61, produced viral titres similar to the well characterised Vesicular Stomatitis Virus pseudotype. Using blinded sera, neutralisation titres were comparable to those using OIE’s gold standard fluorescent antibody virus neutralisation test and assays also revealed cross-neutralisation of EBLV-1, EBLV-2 and CVS-11. This development of an inexpensive and robust high-throughput microassay for the measurement of rabies virus neutralising antibodies in animal and human serum samples will be widely applicable for use in wildlife vaccine campaigns and in human post-vaccination antibody monitoring.