Rabies is a highly fatal zoonotic disease which is primarily caused by rabies virus (RABV) although other members of the genus Lyssavirus can cause rabies as well. As yet， 14 serologically and genetically diverse lyssaviruses have been identified， mostly in bats. To assess the quality of rabies vaccines and immunoglobulin preparations， virus neutralization tests with live RABV are performed in accordance with enhanced biosafety standards. In the present work， a novel neutralization test is presented which takes advantage of a modified vesicular stomatitis virus (VSV) from which the glycoprotein G gene has been deleted and replaced by reporter genes. This single-cycle virus was trans-complemented with RABV envelope glycoprotein. Neutralization of this pseudotype virus with RABV reference serum or immune sera from vaccinated mice showed a strong correlation with the rapid fluorescent focus inhibition test (RFFIT). Importantly， pseudotype viruses containing the envelope glycoproteins of other lyssaviruses were neutralized by reference serum to a significantly lesser extent or were not neutralized at all. Taken together， a pseudotype virus system has been successfully developed which allows the safe， fast， and sensitive detection of neutralizing antibodies directed against different lyssaviruses.