Goldenseal (Hydrastis canadensis L.) is a medicinal forest herb native to Appalachia. Its roots and rhizomes are used as an antimicrobial and for the treatment of intestinal ailments. Three alkaloids–berberine, hydrastine, and canadine–are recognized as the major bioactive constituents in goldenseal. One important postharvest processing step for goldenseal is drying; however, it is not known how drying temperature influences the concentrations of these alkaloids. In this study, pre-emergent (dormant) goldenseal samples were freeze-dried or air-dried at six different temperatures (26.7 to 54.4 °C) to determine the relationship between drying temperature and alkaloid content in the rhizome and roots. High performance liquid chromatography analysis showed that berberine and hydrastine levels were unaffected by drying temperature, while canadine levels decreased as temperature increased (0.55% w/w on average when samples were freeze-dried, down to 0.27% w/w on average when dried at 54.4 °C). While canadine is the least abundant alkaloid of the three, it is known to have key antibacterial properties. Developing a more standardized drying protocol for goldenseal could lead to a more predictable phytochemical profile.