Here, we also found that replacement of the cytoplasmic domain of HN with VCT enhanced the PNU112455A infection efficiency and stabilization of the NDV-pseudotyped HIV-Luc virus, while the infectivity was lost when VCT was added directly to the N-terminus of the HN protein, regardless of which combination of F protein was used. These results differed from those seen in previous research with the Sendai virus where similar replacement and fusion were carried out. These results further suggest that among the paramyxoviruses, their characterizations of packaging lentiviral vectors may differ from each other. For paramyxoviruses, receptor BI-87G3 binding and hemagglutininneurominidase active sites reside in the same region of HN. Therefore, the HI assay is sometimes used for evaluating the neutralizing antibodies that recognize receptor-binging site I. After NDV binding to the receptor on the cells, conformational changes in the envelope proteins will take place. The researches on neutralizing antibodies against HIV-1 have shown that numerous antibodies that recognize regions outside of the receptor-binding site are able to neutralize virus infection. For these neutralizing antibodies, the HI assay would be inadequate. More commonly, neutralizing antibodies against NDV are evaluated by VN tests with native NDV. However, the VN test with native NDV is time-consuming, requiring at least 4 days, and less-objective. In this study, we developed a novel assay to evaluate the neutralizing antibodies using the NDV-pseudotyped HIV-Luc virus. This assay is capable of evaluating all neutralization activities of antibodies through detection of viral entrance into host cells. Compared to currently available VN tests, the NDVpseudotyped HIV-Luc virus-based assay only require two days, and is easily performed in large scale with a luminometer. Our results showed that the NT titers determined with the NDVpseudotyped HIV-Luc virus-based assays were around 4 times higher than those with conventional VN tests. This reflects on the greater sensitivity for detecting NT titers using our established luciferase-based assay compared with performing microscopic observations of CPE using the VN assay.