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Dengue virus (DENV) infection is a worsening global health problem. The plaque reduction neutralization test (PRNT) is currently considered to be the “gold standard” to characterize and quantify circulating levels of anti-DENV neutralizing antibody (NAb). Many variations of the PRNT are currently in use and neither the assay nor its performance conditions have been standardized or harmonized between laboratories. We used a well-characterized panel of acute and late convalescent follow-up sera samples from children experiencing primary and secondary DENV infections to evaluate the performance of the dengue PRNT under a variety of testing conditions. Investigators varied cell type, control virus passage, and the use of complement across multiple assay runs of the same sample panel. Our findings indicate wide variation in PRNT titer results in response to varied testing conditions.