Abstract: To establish a high performance liquid chromatography(HPLC)method to determine the content of bacteriostats in the ocular extractives eye drops, Diamonsil C₁₈(4. 6 mm×250 mm, 5 μm)column was used, with gradient elusion by 1% triethylamine solution(pH 3. 0)(mobile phase A)and methanol(mobile phase B). The detection wavelength was 256 nm; the column temperature was 40 °C; and the flow rate was 1. 0 mL/min. Under these conditions, the three bacteriostats of methylparaben, ethylparoben and chlorhexidine acetate showed good resolution. The bacteriostats exhibited good linear relationship between the peak area and the concentration in the concentration range of 0. 1- 80 μg/mL(r> 0. 999 1). The recoveries were from 97. 2% to 104. 1%, and the RSD was 0. 8% to 1. 2%. The content of bacteriostats in all the five batches of ocular extractives eye drops was less than 10% of the prescription amount. It was found that the activated carbon used in the production process had strong adsorption effect on the bacteriostat, and that the lower the temperature and the higher the concentration of activated carbon, the stronger the adsorption of bacteriostatic agent. The adsorption capacity of activated carbon for different bacteriostats is: chlorhexidine acetate > ethylparoben > methylparaben. The results showed that the established HPLC method was easy to operate with high sensitivity and good repeatability. It can be used to determine the content of bacteriostat in ocular extractives eye drops quickly and accurately. In addition, this study reveals for the first time the effect of impurity removal process on bacteriostat in the production of ocular extractives eye drops. It is not suitable to use activated carbon to remove impurities before adding parabens and chlorhexidine acetate bacteriostats. The current work provides a new guiding basis for the monitoring and improvement of the quality of ocular extractives eye drops.