We report the first known instance of the clinical use of 99mTc-mebrofenin hepatobiliary scintigraphy (HBS) for the optimization of radiotherapy treatment planning and for the follow-up of acute toxicity in a patient undergoing stereotactic body radiation therapy for hepatocellular carcinoma. In our experience, HBS allowed the identification and the sparing of more functioning liver areas, thus potentially reducing the risk of radiation-induced liver toxicity.