Yellow fever (YF) is a mosquito-borne viral disease that is endemic to sub-Saharan Africa and tropical South America. Although there is a safe and effective vaccine that has been available for over 75 years, YF remains a public health problem in endemic areas and areas with sporadic YF transmission. Immune responses elicited by natural infection with yellow fever virus and vaccination are marked by production of neutralizing antibodies. Serological cross-reactivity exhibited by flaviviruses poses challenges to the diagnostic tests for detection of YF-specific antibodies. The most specific plaque reduction neutralization test (PRNT) is considered the “gold standard” test for detecting and measuring the neutralizing antibodies. This study was undertaken to develop and validate PRNT test for measurement of YF specific neutralizing antibodies. We validated PRNT test using different parameters including specificity, linearity, precision, accuracy and robustness. The YF PRNT assay was shown to be specific, robustness, precise and accurate. Thus, we have proven suitability of this assay for evaluation of neutralizing antibodies in response to YF vaccination as well as for diagnostic purpose in case of natural infections.