AbstractA novel virus‐like particle (VLP)‐based multivalent recombinant human papillomavirus (HPV) vaccine was developed and evaluated in human, including 14 HPV‐type specific VLP antigens (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The pseudovirus‐based neutralizing assay (PBNA) method is widely used for immunogenicity assessment of HPV vaccine in clinical trials. However, as many as 14 antigen‐specific antibody levels need be determined, PBNA is, for many reasons, challenging and time‐consuming. In this study, we developed a Luminex immunological assay (LIA) and a competitive Luminex immunological assay (cLIA). These methods increase the throughput, reproducibility and precision, as well as reduce the complexity. All assay parameters showed good characteristics in the validation of both methods, benefiting from highly purified and structurally correct VLPs, high specific antibodies, standard VLP‐microspheres and PE‐mAbs conjugating process, adequate assay development and stable system. Validation data support the use of both methods for immunogenicity assessment in clinical trials. LIA showed higher sensitivity than cLIA, and due to limited epitopes of mAb, cLIA detected lower antibody responses, and therefore, fewer antibodies. This work not only supports clinical trials of 14‐valent HPV vaccines more efficiently and reliably, but also provides a set of validation strategies and usable standards for general vaccine immunogenicity testing.