Verbascum nigrum L. (dark mullein) is known to be a rich source of bioactive secondary metabolites, including high value iridoid and phenylethanoid glycosides, hence the development of alternative approaches for their sustainable supply is highly relevant. Here we report the induction of a transformed root culture of V. nigrum by applying sonication assisted Agrobacterium rhizogenes-mediated genetic transformation. Further, nuclear magnetic resonance (NMR)-based metabolomics approach has been successfully applied to study metabolic differences of the V. nigrum mother plant and related transformed roots. ¹H NMR fingerprinting in combination with 2D NMR and multivariate data analysis revealed that the most abundant molecule in the hairy root culture is glutamine, which is not found in the mother plant tissue. These findings provide important insights on the metabolic alterations as a result of the transformation of the host plant genome and development of so-called hairy roots.