Objective：To establish a reverse-phase high performance liquid chromatographic（RP-HPLC） assay for the determination of vacomycin levels in rabbits aqueous humor. Methods：The vancomycin extracted by the sedimentation with 10% ZnSO4 was detected through RP-HPLC. The analytical column was a C8 column. Twenty microlitres of samples were injected directly into the column and were eluted with a gradient consisting of methanol（A） and acetic acid（B）. The sample was collected and analyzed after nano-vancomycin eyedrops administered. Results：The drug peak was separated well without interference from the aqueous humor. The peak area and concentration of the drug in aqueous humor were linearly correlated within the range of 0.5-20 μg／ml, with a correlation coefficient of 0.9990. The average recovery rate of the samples of 1，10，18 μg／ml was（98.67０ ± 0.321）%, and the precision and accuracy of the inter- and intra-day was（3.40 ± 1.30）%，（3.80 ± 2.20）% respectively. The concentration of vancomycin in rabbits aqueous humor at 120 min after nano-vancomycin eye drops administered was 0.83 ± 0.05 μg／ml. Conclusion：RP-HPLC assay is simple and sensitive, which may be used in ocular pharmacokinetic studies of vancomycin.