High frequency shoot regeneration and evaluation of product synthesis at various stages in Psoralea corylifolia were investigated. In vitro regenerated shoots were induced from germinated seedling on MS medium supplemented with 2, 4, 8, 20 and 40 μM of thidiazuron and N 6–benzylaminopurine. The results revealed that optimum concentrations of thidiazuron (8 μM) into the medium increased shoot regeneration frequency. Root differentiation was achieved from regenerated shoots on growth regulator free MS medium with frequency of 91.2% and mean number of 4.5 roots per shoot. High concentrations of Indole-3-aceticacid (IAA) into the rooting medium resulted in slow growth. Regenerated shoots and roots enhanced isoflavones production compared to field grown plants. A reverse phase high performance liquid chromatography analysis revealed that in vitro regenerated shoots accumulated 0.85% dry wt of daidzein and 0.06% dry wt of genistein. Maximum daidzein (1.23% dry wt) and genistein (0.38% dry wt) were accumulated by roots which obtained from regenerated shoots, which is 6.3-fold more daidzein and 77-fold more genistein respectively than field grown plants. The regeneration protocol developed successfully in this study showed the possibility for rapid propagation of P. corylifolia and enhanced isoflavones production.