Asteracantha longifolia (Linn.), also known as Hygrophila auriculata, is an important medicinal plant in Ayurvedic and Unani medicine. This study evaluated the toxicity of ethanolic seed extracts in rats. Acute toxicity studies administered single doses of 5, 50, 300, and 2000 mg/kg and observed animals for 14 days. Subacute studies involved oral doses of 100, 200, and 400 mg/kg for 28 days. Animals were monitored for general behavior, body weight, food and water intake, and signs of morbidity or mortality. No significant toxicity, mortality, or adverse effects were observed. The results indicate the extract is safe in both acute and subacute studies, supporting further research in animals and humans for therapeutic efficacy. © 2020 Elsevier B.V., All rights reserved.