ABSTRACT The plaque reduction neutralization test (PRNT) is a specific serological test used to identify and confirm arbovirus infection in diagnostic laboratories and monitor immunological protection in vaccine recipients. Wild-type (wt) viruses used in the PRNT may be difficult to grow and plaque titrate, such as the dengue viruses (DENV), and/or may require biosafety level 3 (BSL3) containment, such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). These requirements preclude their use in diagnostic laboratories with only BSL2 capacity. In addition, wt JEV falls under the jurisdiction of the select-agent program and can be used only in approved laboratories. The chimeric vaccine viruses ChimeriVax-WNV and -SLEV have previously been shown to elicit antibody reactivity comparable to that of parental wt WNV and SLEV. ChimeriVax viruses provide advantages for PRNT, as follows: they grow more rapidly than most wt flaviviruses, produce large plaques, require BSL2 conditions, and are not under select-agent restrictions. We evaluated the ChimeriVax-DENV serotype 1 (DENV1), -DENV2, -DENV3, -DENV4, and -JEV for use in PRNT on sera from DENV- and JEV-infected patients and from JEV vaccine recipients. Serostatus agreement was 100% between the ChimeriVax-DENV serotypes and wt prototype DENV and 97% overall with ChimeriVax-JEV compared to prototype Nakayama JEV, 92% in a subgroup of JEV vaccine recipients, and 100% in serum from encephalitis patients naturally infected with JEV. ChimeriVax-DENV and -JEV plaque phenotype and BSL2 requirements, combined with sensitive and specific reactivity, make them good substitutes for wt DENV and JEV in PRNT in public health diagnostic laboratories.