The validation of a bioanalytical method is essential to ascertain its suitability for a specific purpose and ensure the accuracy of its analytical outcomes, particularly within the pharmaceutical industry. The pseudovirion-based neutralization assay (PBNA) is considered the gold standard for detecting and quantifying neutralizing antibodies against human papillomavirus in the development of the disease-prevent vaccines. This paper introduces a modified high-throughput triple-color PBNA method designed to simultaneously detect the neutralizing antibodies against as many as 14 HPV types. The primary objective is to present a comprehensive validation of the triple-color PBNA method applicable to general vaccine immunogenicity assays. The results demonstrate good specificity, accuracy, precision, linearity, robustness, and applicability of the method. This innovative triple-color PBNA provides a high-throughput approach for large-scale immunogenicity assessments that vaccine developers might encounter, overcoming limitations of labor-intensive and complex nature associated with traditional neutralization assays. The research establishes a robust foundation and good guiding example for evaluating vaccine responses in both preclinical and clinical phases, offering valuable insights to the field.