Objective：To establish a practical HPLC method to determine the concentration of methazolamide in aqueous humor of rabbit eyes for the research of methazolamide drug delivery system. Method：Protein was deposited by 10%（V／V） ｔrichloroacetic acid. Methazolamide was extracted from aqueous humor by using C18 column and mobile phase of methanol ∶ water（25 ∶ 75，V／V）. The column temperature was maintained at 40℃, methazolamide was detected at 290 nm, and the flow rate was 1.0 ml／min. Results：A good linear relationship was obtained between the concentration of methazolamide from 0.05 -滋g／ml to 10 -滋g／ml, r = 0.9982. The Relative recovery and absolute recovery of methazolamide were 101.7% and 82.7%, respectively; and RSD of intra-day and inter-day were less than 10%. Conclusion：The improved method could significantly enhance the detective sensitivity without damage to the column and increase the accuracy. The procedure was simple, sensitive, and suitable for determination of methazolamide in aqueous humor with good practicability.