Houttuynia cordata Thunb. (HC), a perennial plant distributed mainly in the tropics and subtropics regions, has been widely used as a folk medicine in Asian countries such as Vietnam. Nevertheless, limited studies have reported the pharmacognostical standardization and toxicity of the Vietnamese HC. Therefore, this study collected, identified, extracted, and in-vivo toxicological tested various HC samples at four locations, representing the whole Vietnam, including Hanoi (the Northern area), Dak Lak (the Western area), Bien Hoa (the Middle area), and Long An (the Southern area). All plant samples satisfied the quality requirements according to the standards of the Vietnamese Pharmacopoeia V and Hong Kong Pharmacopoeia. Next, a standard extraction and preparation process of HC was developed with an extraction solvent of 70% ethanol, concentrated under reduced pressure to a density of 1.16 g/mL, and spray-dried with excipients of Syloid244FP:lactose (1:2 w/w) to obtain the crude ethanolic extract of HC with optimal recovery efficiency (71.35%). Finally, the crude HC extract was evaluated its acute toxicity in rats at a dose of 50 g/kg body weight, and sub-acute toxicity in rabbits at a dose of up to 1.5 g/kg/day. No potential toxicity was noted in both settings. Conclusively, the Vietnamese HC extract, which was standardized and possessed safeness in both rat and rabbit animal models, could be further investigated to become a pharmaceutical agent in the future.