The incidence of liver diseases, such as nonalcoholic fatty liver disease and drug-induced liver injury, continues to rise and is one of the leading causes of acute hepatitis. Current trends suggest that these types of conditions will increase in the coming years. There are few drugs available for the prevention or treatment of hepatic diseases, and there is a growing need for the development of safe hepatoprotective agents. The medicinal plant, Turnera diffusa, has many ethnopharmacological uses, one of which is the production of a flavonoid named hepatodamianol, which is the principal component responsible for this plant's hepatoprotective properties. In the present study, we describe the development and standardization of an active extract obtained from T. diffusa. We conducted nuclear magnetic resonance spectroscopy to identify hepatodamianol unambiguously in each sample. Using this extract, hepatoprotection could be demonstrated in vivo for the first time. The hepatoprotective effect did not display a significant difference in vivo when compared with silymarin used as a positive control at the same doses. Implementation of quality criteria used for standardization, such as flavonoid and hepatodamianol content, hepatoprotective activity, and absence of residual solvents, will allow future preclinical trials with this herbal drug.