We have established an efficient protocol for plant regeneration and production of secondary metabolites in hairy root culture of Centaurium erythraea Rafn. Because the hairy roots and regenerated plants produce bitter secoiridoid glucosides and xanthones similar to the plants in nature, the use of in vitro cultures as an alternative source of their production is feasible. This chapter describes a protocol for the induction of adventitious shoots and transgenic plants from hairy root cultures of C. erythraea and their phytochemical analysis.