A high throughput liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of berberine and hydrastine in human serum, after oral administration of goldenseal (Hydrastis canadensis L.), was developed using simple acetonitrile treatment of serum samples. Noscapine served as the internal standard. Lower limit of quantification for both analytes was 0.1 ng mL(-1) using positive ion electrospray tandem mass spectrometry (MS/MS). The intra-day (n=5) accuracy and precision of the method for hydrastine was 82+/-8.8%, 97.9+/-2.4% and 96.2+/-3.3%, respectively. The inter-day (n=4) accuracy and precision for hydrastine was 90.0+/-15.17%, 99.9+/-7.1% and 98+/-6.54%, respectively. For berberine quantitation intra-day accuracy and precision was 96.0+/-8.4%, 92.5+/-4.7% and 94.4+/-3.7%, respectively. The respective values for inter-day quantitation were 91.0+/-8.4%, 94.3+/-4.7% and 94.4+/-3.7%. The analytical recovery for hydrastine was 82.4-96.2% and for berberine it was 94.4-96.0%. The analytes and noscapine were stable for 24h at room temperature (CV 5-10%). Matrix ion effects were studied by post-column infusion of hydrastine and berberine, calculation of calibration curve slope precision was obtained using serum from five different subjects, and by comparison of the response of methanol standards and extracted serum samples. The method was further validated by determination of serum pharmacokinetics of hydrastine and berberine after administration of a single oral dose of goldenseal extract containing 77 mg of hydrastine and 132 mg of berberine.