Japanese encephalitis virus is a common cause of viral encephalitis in Asia with an estimated 45,000 cases annually. It causes significant morbidity and mortality. It is transmitted primarily by Culex mosquitoes between birds and animals, while man is thought to be an accidental, dead-end host. Since dengue is also prevalent usually in Japanese encephalitis-endemic areas, all Japanese encephalitis positive sera must be confirmed by detecting Japanese encephalitis specific neutralizing antibodies. The plaque reduction neutralization test is the gold standard for detecting and quantifying Japanese encephalitis neutralizing antibodies. This test, however, takes about a week and is carried out in 6 or 24-well plates, which limits its usage for large-scale screening. A simplified assay was developed for the detection and quantification of Japanese encephalitis neutralizing antibodies. The assay, which is carried out in 96-well plates, would be suitable for use in the mass screening of the population's immunity level as well as for use in vaccine efficacy studies.