Technical Abstract: White snakeroot (Ageratina altissima) and rayless goldenrod (Isocoma pluriflora) can cause “trembles” and “milk sickness” in livestock and humans, respectively. Tremetol, a complex mixture of sterols and derivatives of methyl ketone benzofuran has been extracted from white snakeroot and rayless goldenrod and is reported to be the toxic substance in plant material. In this study, the three major benzofuran ketones, tremetone, dehydrotremetone, and 3-oxyangeloyl-tremetone were isolated from rayless goldenrod. Using these compounds as standards, a quantitative HPLC method was developed to measure these compounds in white snakeroot and rayless goldenrod. Concentrations of tremetone, dehydrotremetone, and 3-oxyangeloyl-tremetone were found to vary considerably among the different white snakeroot and rayless goldenrod plant collections.