Berberine is a safe and effective anti-arrhythmic drug. We do not clearly know about the dose-effect relationship. Besides, berberine is not fit for administration by i.m. and i.v.. Therefore, it is necessary to build a PD model of berberine, study its pharmacodynamics parameters in vivo, and provide a concrete criteria in the clinical usage. In this article, we set up a method for determination of concentration of berberine in human serum by RP-HPLC with Lichrosorb RP-18 column (250 mm x 4 mm) and mobile phase of MeOH containing 0.15% triethylamine. The content of berberine was determined by the external standard method with UV detector at 347 nm. The linear range was 0.2-2 mg/L (r = 0.9996). The detectable limit was 2.55 ng. The average recovery was 90.73% and the coefficients of variation were below 8.0%. The method is sensitive and accurate.