We used HPLC method for determination of berberine bisulfate and trimethoprimin compound berberine injection,The mobile phase consisted of a minxture of methanol ammoni-um acetate solution(1 mol/L,pH 5.5)(80:20).The flow rate was 1 ml/min。Wavelength was de-termined 265 nm。Phaenacetine was selected as internal standard. A ODS-C18 column was used。The standard curve of berberine bisulfate was linear in the range from 0. 10～0. 30 mg/ml. Theaverage recovery was 101.66%,RSD=1. 6%(n=6). The standard curve of trimethoprim was lin-ear in the range from 0. 25～1. 25 mg/ml.The average recovery was 101. 42%,RSD=1. 19%(n=6)。