ETHNOPHARMACOLOGICAL RELEVANCE: Artemisia roxburghiana Wall. ex Besser is a well-known remedy for treating fever and diabetes. Natives of the Himalayan region use it to treat malaria, dysentery, rheumatism, and viral hepatitis. AIM: The current investigation aimed to evaluate the phytochemical profile, acute and sub-acute toxicity of the aqueous leaf extract of Artemisia roxburghiana. METHODS: Liquid chromatography coupled with mass spectrometry determined the phytochemical profile. The potential toxicity of A. roxburghiana was evaluated by executing acute and sub-acute toxicity according to guidelines 423 and 407 of OECD. A single dose of 2 g/kg body weight was gavaged orally in acute toxicity. Animals were observed for memory impairment, depressive/anxiogenic behavior, and mortality for 14 days. In sub-acute toxicity, 200, 400, and 600 mg/kg body weight of extract was given for 28 days. Body weights, relative organ weights, hematological, histological, biochemical parameters, and endogenous antioxidants were assessed and compared with control. RESULTS: LD CONCLUSION: The extract was rich in bioactive compounds with determinate therapeutic benefits. The plant was safe in repeated administration for 28 days which justifies its expansive use in traditional medicine systems.