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Artemisinin and its derivatives are still one of the most effective drugs for the treatment of malaria. Artemisia pallens commonly known as Dhavanam, is an aromatic herb belonging to the family Asteraceae. Increasing the artemisinin content of A. pallens by genetic engineering would improve the availability of this much needed drug. In the present study, Agrobacterium rhizogenes (strain NCIM 5140) mediated genetic transformation of Artemisia pallens were carried out for hairy root induction. The effect of different media (Half MS, MS, MS along with BAP 0.5 mg/l and MS along with Kinetin 0.5 mg/l) and type of explants (leaf and stem) on hairy root induction and culture were also studied. Maximum transformation efficiency (70.0 %) was observed in case of stem explants when it was co-cultivated with Agrobacterium rhizogenes and kept on half strength MS media. Artesunate is a derivative of artemisinin, was quantified using HPLC from dried aerial extract and hairy roots. The content of artesunate in hairy roots was increased up to twofold as compared to aerial part of Artemisia pallens. The maximum amount of artesunate found in hairy roots was 5.62 ± 0.16 μg/g of dry weight. Apart from artesunate the other phytochemicals like alkaloids, polyphenols, and flavonoids are important because they impart the medicinal properties in this plant. Therefore, we have also quantified total alkaloids, flavonoids and polyphenolic content in the aerial part of the plants. The total alkaloids and flavonoids content were found 1.72 ± 0.00 mg/g dry weight in aqueous extract and 3.8 ± 0.00 mg/g in methanolic extract in terms of colchicine and rutin equivalents, respectively. Similarly, total phenolic content is 3.70 ± 0.01 mg/g in ethanolic extract in terms of tannic acid equivalent.