Abstract: OBJECTIVE To establish a quantitative analysis method of the high molecular weight proteins in insulin degludec. METHODS The column was Insulin HMWP column(7.8 mm×300 mm, 10 μm). The mobile phase was acetic acid-isopropanol- water (4：3：10). The flow rate was 0.5 mL·min^-1. The detection wavelength was set at 280 nm. The high molecular weight proteins were separated and accurately determined by size exclusion chromatography. RESULTS The detection limit and quantitative detection limit were 0.18, 0.47 μg, respectively. The RSD of instrument precision test was 0.51%. The concentration of insulin degludec at the range of 0.125-4.0 mg·mL^-1 showed good linear relationship to the peak area, of which the regression equation was Y=20.864 1X-0.125 9, with R²=1.000 0. The resolution of main peak and molecular protein peak was >2.0. CONCLUSION The established method is simple, sensitive and can effectively quantitatively analyze the high molecular protein of insulin degludec.