Abstract: OBJECTIVE To establish a quantitative assay method for determining the titer of HPV16 L2E6E7 vaccine during its development and manufacturing. METHODS By using a conventional method, rabbit anti-HPV16 L2E6E7 polyclonal antibodies were generated and used together with a commercially available mouse anti-HPV16 E7 monoclonal antibody to establish a sandwich ELISA method for HPV16 L2E6E7 vaccine. The linearity, sensitivity, and specificity of the method were evaluated. RESULTS A specific and sensitive ELISA method for assaying HPV16 L2E6E7 vaccine quantitatively was developed. Good linearity was shown within the detection range of 19.53-1 250 ng·mL^-1(R²>0.99), and the recovery rate was 90%-110%. The method showed strong sensitivity and high specificity with no interference by host cell proteins of E. coli. CONCLUSION A quantitative ELISA method is established for HPV16 L2E6E7 vaccine. It offers a useful technical tool for quality control of the vaccine during its fermentation process.