Periwinkle (Catharanthus roseus L.) is one of the most important medicinal plants that is commonly used for its anticancer alkaloids. In this investigation, the effects of different tryptophan concentrations (0, 150, 250 and 350 mg/l) on in vitro regeneration conditions were studied. The regeneration of different explants on different tryptophan-free media compositions showed that the best media for growing nodal segments explants was MS medium containing (BAP, 0.5 mg/l)+(NAA, 1 mg/l). However, the rooting media composition was determined as half MS medium supplemented with (IBA, 0.1 mg/l). After adding tryptophan into this optimal medium, the maximum shooting and rooting percentages were obtained with 250 and 350 mg/l tryptophan, respectively. Furthermore, the measurement of shoot and root length, dry and fresh weight of shoot, fresh weight of root, chlorophyll a and carotenoid content and also, soluble sugars content of shoot and root, revealed that the best medium was in the presence of 350 mg/l tryptophan, while the maximum amount of root dry weight and chlorophyll b were obtained by 250 mg/l tryptophan containing medium. Finally, the analysis of total proteins content of shoot and root showed a significant difference between all groups and higher content of proteins for shoots and roots was observed in 250 mg/l and 350 mg/l tryptophan, respectively.