A HPLC method for determination of preservatives benzalkonium chloride in pranoprofen eye drops was established.A C18 column (4.6 × 250 mm, 5 μm) was used with the mobile phase of 0.02 mol/L sodium heptanesulfonate solution (0.1% triethylamine adjusted pH to 3.45 ± 0.1 with phosphoric acid)-acetonitrile (35:65), at the detection wavelength of 208 nm, at the flow of 1.5 mL/min and the column temperature of 40°.The content of C12 in the benzalkonium chloride was the highest, and the contents of C14 and C16 in the benzalkonium chloride didn't reach the limit of detection.The peaks of C12, pranoprofen and blank materials can achieve an effective separationThe HPLC method showed sensitive, accurate, and reproducible, and can be effective in the content determination of preservatives benzalkonium chloride in pranoprofen eye drops.