The eradication of smallpox, a historic triumph in global public health, was accomplished without a complete conception of the mechanisms underlying vaccine-induced protection. Contemporary concerns regarding potential bioterrorism threats and the possibility of smallpox reemergence have spurred research efforts toward developing third-generation vaccines capable of effectively neutralizing the variola virus. Clinical trials for a third-generation smallpox vaccine (KVAC103) are underway to obtain licensure. As a surrogate marker for efficacy, vaccinia virus (VACV) antibody levels can be assessed using the plaque reduction neutralization test (PRNT). In the current study, the PRNT methodology underwent comprehensive development, optimization, and validation in strict adherence to the guidelines for bioanalytical test methods. The VACV PRNT50 was optimized to include the working virus concentration (4 × 102 plaque-forming units/mL), virus-serum neutralization time (60 min), concentration of carboxymethylcellulose sodium salt overlay (1 %), and days of incubation post infection (3 days). Using human serum samples from individuals administered the second-generation smallpox vaccine (CJ-50300), the VACV PRNT50 (cut-off point, 22.58), based on the receiver-operating characteristic curve (area under the curve = 0.9859) and sensitivity and specificity assays, exhibited favorable outcomes, showing 93.75 % specificity (95 % confidence interval [CI], 71.67-99.68 %) and 93.55 % sensitivity (95 % CI, 79.28-98.85 %) against the VACV strain Western Reserve. The validation process encompassed crucial parameters, including intra-assay and inter-assay precision, robustness, dilution linearity, and the lower limit of quantification. The VACV PRNT50 exhibited high accuracy and 100 % intra-assay and inter-assay precision across various ND50 titers (high, middle, and low). Overall, the PRNT was validated as a reliable tool for measuring VACV-neutralizing antibodies and evaluating the effectiveness of new smallpox vaccinations in human serum samples.