OBJECTIVE： To establish a method for the simultaneous determination of chloramphenicol， prednisolone acetate and chloramphenicol glycols in Compound chloramphenicol eye drop. METHODS： HPLC was performed on the column of Dionex Acclaim C18 with mobile phase of 0.1％ Heptane sodium sulfonate solution-glacial acetic acid-acetonitrile-methanol （60 ∶ 0.2 ∶ 30 ∶ 10，V/V/V/V） at a flow rate of 1.0 mL/min， detection wavelength was 271 nm， column temperature was 30 ℃， and the injection volume was 10 μL. RESULTS： The linear range was 11.73-703.71 μg/mL for chloramphenicol （r＝0.999 8）， 12.40-744.06 μg/mL for prednisolone acetate （r＝0.999 8） and 1.03-62.03 μg/mL for chloramphenicol glycols （r＝0.999 8）； limit of quantification of chloramphenicol glycols was 0.01 μg/mL； RSDs of precision and stability tests were lower than 2.0％； RSDs of reproducibility test for chloramphenicol and prednisolone acetate were lower than 2.0％， RSD of chloramphenicol glycols was 4.5％； recoveries were 100.6％-102.7％（RSD＝0.60％，n＝9）， 97.5％-100.3％ （RSD＝0.80％，n＝9）， 100.0％-103.2％ （RSD＝1.10％，n＝9）， respectively. CONCLUSIONS： The method is accurate， simple， and can be used for the simultaneous determination of chloramphenicol， prednisolone acetate and chloramphenicol glycols in Compound chloramphenicol eye drop.