OBJECTIVE To establish a method for determining the concentration of dexamethasone phosphate in the perilymph of guinea pig. METHODS Typmpanic cavity was infused with dexamethasone phosphate solution,the perilymph of the scala tympani was sampled at 30 min after an administrations.Dexamethasone phosphate in the samples was assayed by HPLC.A separation was performed on a ZORBAX Eclipse XDB-C_(18) column with 7.56 mmol·L~(-1) ammonium sulfate-acetonitrile(70∶30) as mobile phase at the flow rate of 1.0 mL·min~(-1).The temperature of column was at 20 ℃,the wavelength of detection was 239 nm.RESULTS The calibration curve was linear within the concentration range of 0.10～4.8 mg·L~(-1)(r=0.999 8),the limit of detection was 6.25 μg·L~(-1),the RSD values of interday and intraday ralidation were 2.17% and 3.25%.The concentration of dexamethasone phosphate in the perilymph of the scala tympani was 2.20 mg·L~(-1) at 30 min after administration.CONCLUSION The method appeares to be simple,accurate,reproducible and can be used to determine the concentration of dexamethasone phosphate in perilymph.