The content of benzalkonium chloride in Dextran 70 and glycerol eye drops was determined by HPLC.The chromatog. conditions were as follows: the column was Kromasil C18 (250 mm × 4.6 mm, 5 μm) column; the mobile phase consisted of acetonitrile-0.03 mol/L-1 sodium dihydrogen phosphate (pH 7.0) (43:57); the flow rate was 2.0 mL/min; the column temperature was 35°; the detection wavelength was at 214 nm; the injection volume was 20 μL; the separable degree of benzalkonium chloride between C12, C14, C16, should meet rules.The calibration curve of benzalkonium chloride ranged from 1.28-3.00 μg had good linearity with peak area (r = 0.9999, n = 7).The recovery (n=6) was 100.9%; RSD=0.9%.The method is simple, fast, and accurate, with high sensitivity and good repeatability, and can be used for content determination of benzalkonium chloride in Dextran 70 and glycerol eye drops.