In vitro regeneration of Ruta graveolens L. was carried out using three different explants (shoot tips, lateral buds and leaves) with three medium protocols to determine the best explants, which was shoot tips, linked with the optimum medium, which was MS+ 0.5 mg/l BAP + 0.3 mg/l NAA, for the high potentiality of shoot formation. To induce mutation, the best explant was treated with four doses of gamma radiation 10,20,30,40 Gy. Radiation levels affected shoot length and leaves number, where shoot length and leaves number increased by 10 Gy dose. Finally, rutin was extracted from shoots of field mother plant, in vitro plantlets and irradiated plants and determined using U.V. spectrophotometer. Plantlets irradiated by 10 Gy contained the highest amount of total rutin, (rutin in DW), whereas the lowest amount of total rutin was at 40 Gy. The in vivo unirradiated plantlets exhibited total rutin greater than those of 40 Gy irradiated plantlets, thus gamma radiation can affect the plant phenotype and the production of rutin.