Abstract: Objective To establish an amino acid assay for the determination of β-lactoglobulin in Anti-HPV biological protein dressing. Methods Under acidic conditions, β-lactoglobulin is hydrolyzed into free amino acids, separated by cation exchange chromatography, and derivatived after ninhydrin column. The chromatogram at 570 nm is collected. The content of β-lactoglobulin in the sample is indirectly determined by measuring the lysine content obtained by hydrolysis. Results β-lactoglobulin has a good linear relationship in the concentration range of 77.28~309.12 μg/mL (y=5.060x+4.278, r=0.999 7); The recovery rate (n=9) is 100.06%. Conclusion The method is simple, specific, accurate and reproducible, which is suitable for the quantitative analysis of β-lactoglobulin in anti-HPV biological protein dressing.