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Reversed-phase high-performance liquid chromatography (RP-HPLC) method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column (250 mm×4.6 mm×5μm) and Shiseido CAPCELL PAK SG 300 C18 column (250 mm× 4.6 mm×5 μm) were used in the experiment. Phase A was composed of 0.1% (V/V) trifluoroacetic acid (TFA) in ultrapure water and Phase B (organic phase) was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.