The disclosure relates to the development of improved methods for quantifying antigen in a vaccine composition in the absence of available antigen standards. More specifically, the disclosure provides fast and robust methods of separating antigens from vaccine compositions, comprising the steps of solubilizing antigen without detergent and without alkylation, using acidification to prevent antigen subtypes from binding again, isolating antigen subtypes with chromatography, and quantifying the eluted antigen with amino acid analysis. The methods of the disclosure are applicable for use with a variety of antigens, thereby providing an improved method in the art of vaccine manufacturing to date.
