Human papillomavirus type 52 (HPV 52) infection is epidemiologically predominant in low-middle income countries in South-East Asia and remains a threat for global health. This study aims to assess the immunogenicity of prophylactic vaccine candidate formulated from HPV 52 L1 virus-like particles (VLPs). A codon-optimized and N-terminally truncated HPV 52 L1 gene was cloned using pHIPZ4 plasmid and expressed in Hansenula polymorpha. Protein purification was performed by one-step cation exchange chromatography and was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The VLPs formation was confirmed by transmission electron microscopy (TEM) prior to formulation using AlPO4 adjuvant and immunization of BALB/c mouse. The mouse sera were analyzed by enzyme-linked immunosorbent assay (ELISA), and green fluorescent protein (GFP) pseudovirion-based neutralization assay was performed for immunogenicity study. The purified HPV 52 L1 protein was successfully assembled into VLPs and showed a recovery yield of 51.76 %. The L1 antigen demonstrated a high antibody titer production, suggesting that the vaccine formulation induced humoral immune response in mouse model. Moreover, the antibody from mouse sera were able to neutralize HPV 52 pseudovirus infections in human embryonic kidney 293 (HEK293) cells. These findings suggest that the production of HPV 52 L1 VLPs using H. polymorpha expression system induces immune response, thus potentially can be developed as alternative HPV vaccine. © 2025 International Alliance for Biological Standardization