Hispanon and leoheteronin B were isolated from Leonurus heterophyllus and further established as reference substances under the guide of Asean-USP and ISO 34. By the developed method, which was validated to show specific, accurate and precise, the contents of hispanon ahd leoheteronin B were 96.68 percent and 100.00 percent, respectively. That meant they promised to serve as references in quality control of medicinal plant materials and herbal products.