Flaviviruses represent a major global health concern, with yellow fever virus (YFV) and dengue virus (DENV) among the most studied members of the Orthoflavivirus genus. Transmitted by Aedes mosquitoes, these viruses are responsible for severe disease outbreaks worldwide. While YFV benefits from an effective vaccine, it remains a threat in endemic regions. Dengue, with its four distinct serotypes, continues to complicate vaccine development and disease management. Plaque assays and the plaque reduction neutralization test (PRNT) are key tools for assessing viral replication and immune responses. PRNT50, commonly used in postvaccination studies, quantifies the serum dilution needed to neutralize 50% of the virus, providing insights into vaccine-induced immunity. In contrast, PRNT90, with a higher neutralization threshold, is more appropriate for diagnostics and epidemiological surveillance, offering a stringent measure of past exposure and protection. This chapter focuses on the application of PRNT in yellow fever (YF) vaccine research, highlighting PRNT50 for evaluating immunogenicity and PRNT90 for population-level immunity assessments. We present findings from a core study validating the PRNT assay, followed by clinical research supporting fractional dosing strategies in various populations, including children and immunocompromised individuals. Together, these studies underscore the value of PRNT in optimizing vaccine use and advancing public health strategies against Orthoflavivirus infections.