At present, the haemagglutination inhibition and the virus-neutralization assays are available for the detection of neutralizing Verdinexor antibodies against NDV. While the HI assay is able to measure only the neutralizing antibodies against the receptor-binding site and has limitations in terms of its low sensitivity and high incidence of false-positives, the conventionally used VN assay is labor-intensive, time-consuming, and less objective, making it unsuitable for large scale evaluation of neutralizing antibodies. Pseudovirus-based neutralization assays, however, have been proven to be a rapid, sensitive, and specific high-throughput system for the evaluation of neutralizing antibodies and antiviral drug discovery. The pseudovirus backbone generally carries a reporter gene, such as luciferase, in which the neutralizing ability of antibodies can be easily quantified. Pseudotyped viral particles by heterologous viral glycoproteins have been described for several viruses, including influenza virus, SARS CGP36742 coronavirus, Sendai virus and hepatitis C virus. However, pseudotyped viruses with the NDV envelope glycoproteins HN and F have not yet been reported. Here, we report on the successful production of HN and F-pseudotyped HIV-Luc viral particles and the factors impacting the infection efficiency of NDV-pseudoviruses. We further established a novel neutralization assay with the NDV-pseudotyped HIV-Luc viruses to evaluate neutralizing antibodies against NDV. Pseudotyping provides the ability to introduce proteins from different sources into a viral vector shell. Many pseudotyped vectors have been developed using retroviruses or lentiviruses, such as the Mo-MuLV vector from Moloney murine leukemia virus and HIV-1 vector from the HIV-1 virus. Given that some components essential for viral replication or persistent infection have been deleted from the genome, pseudotyped viruses can only take on a single-cycle of infection. Thus, they are a safe alternative for use in the research lab with extremely virulent viruses. So far, pseudoviruses have been used for the identification of virus receptors, screening and evaluation of neutralizing antibodies, transduction of genes to target cells and exploration of the mechanisms of virus infection.