An isocratic high-performance liquid chromatography (HPLC) method was developed for the analysis of hydrastinine, palmatine, berberine, hydrastine, and canadine, all alkaloid components known to be present in goldenseal root powder. Optimized separation was achieved on a Zorbax Eclipse-XDB column at 30 degreesC using a mobile phase of 10 mM ammonium acetate/acetonitrile (70:30, v/v) at a flow rate of 1.0 mL/min with ultraviolet detection at 235 nm. The method showed linearity for palmatine, berberine, hydrastine, and canadine at approximately 4-400 mug/mL, while hydrastinine was linear at approximately 4-80 mug/mL. Method precision and robustness were investigated. An example of an HPLC analysis of goldenseal root powder extract is presented here also. These studies indicate that the method described here is usable for analysis of goldenseal extracts.