Liquid chromatography coupled to multistage mass spectrometry (LC-MSn) is being used increasingly in pharmaceutical research and for quality control in herbal medicines because of its superior sensitivity and selectivity. In this study, a rapid, high-resolution liquid chromatography-mass spectrometry (LC-MSn) method was developed to separate and identify alkaloids in the root extract of goldenseal, which is one of the 20 most popular herbal supplements used worldwide. In total, 28 alkaloids were separated and characterized including one novel compound and 21 identified, or tentatively identified, for the first time in goldenseal. The current high-resolution LC-MSn method provides a rapid and definitive means of profiling the composition of goldenseal root and will provide a useful tool in understanding the bioactivity of this medicinal plant.