"Hedgehyssop" Gratiola officinalis L. (Scrophulariaceae) is found as an ingredient in homeopathic remedies. Among the active compounds found in G. officinalis, the cucurbitacines constitute a group of triterpenoid substances which are well-known for their bitterness and toxicity. Due to the toxicity of the cucurbitacin's aglycones it becomes necessary to determine the content of the aglycones and glycosides that may be responsible for the pharmacological activity and toxicity in homeopathic tinctures according to the European Pharmacopeia guidelines. In this context a HPLC method was developed for the identification and determination of cucurbitacin E and I in homeopathic mother tinctures. To evaluate the total concentration of the aglycones, cucurbitacin E and I formed after hydrolysis we determined the concentration of both compounds after enzymatic hydrolysis with beta-glucosidase in vitro. Reversed-phase HPLC with a Eurospher C-18 column with precolumn and acetonitrile-water gradient system as the mobile phase proved to be suitable for direct determination of both aglycons, cucurbitacin E and I in Gratiola-mother tinctures. The contents of cucurbitacin E, cucurbitacin 1, cucurbitacin E glycoside and cucurbitacin I glycoside were found as 0.0065%, 0.0031%, 0.0011% and 0.0006%, respectively in Gratiola-mother tincture prepared according to method 2a HAB.