Spilanthes acmella is an important medicinal plant that has been administered as a traditional medicine for years to cure toothaches, stammering, stomatitis and many other ailments. The which resulted in overexploitation and depletion of S.acmella from its natural habitat. The plant and its highly valuable medicinal product, spilanthol are not meeting the industrial demand due to less commercial cultivation. Hence, the present study was carried out with the objective to standardize the induction of hairy roots in S.acmella by genetic transformation with of spilanthol. This study demonstrated the induction and multiplication of hairy roots from nodal segments and leaf explants taken from field grown and in vitro developed S.acmella plants.PCR analysis of hairy root was performed that confirmed the bacterial transformation. S.acmella. The methanolic extract of hairy roots showed significant amount of spilanthol content (0.134%). This study is the first report of quantification of spilanthol content in hairy roots of S.acmella, which offers a great potential for their use as an alternative source for the continuous production of spilanthol