This study investigated the effect of plant growth regulators on the initiation of in vitro culture of Stevia rebaudiana Bertoni. The experiment focused on surface sterilization protocols and the evaluation of explants derived from stem nodes, along with studying the effect of cytokinins (BA and Kin) on shoot regeneration. Surface sterilization with 4% sodium hypochlorite (NaOCI) for 6 minutes showed the highest effectiveness. The explants were cultured on MS medium supplemented with benzyl adenine (BA) at concentration of (0.0, 0.5, 1.0, 1.5, 2.0) mg.l-1 and kinetin (Kin) at concentration of (0.0, 2.0, 4.0, 6.0, 8.0) mg.l-1 during the establishment and multiplication stages.In the establishment stage, the highest response rate (100%) was recorded for the concentration of 1.5 and 0.5 mg.l-1 of BA, and for Kin at a concentration of 4.0 mg.l-1. The highest number of shoots (9.20 shoots. explant -1) and the highest total number of leaves (58.30 leaves. explant -1) were achieved with 1.0 mg.l-1 of BA, while the longest shoot length (7.15 cm) was achieved with 1.5 mg.l-1 of BA.In the multiplication stage (after 8 weeks), treatment with 1.5 mg.l-1 of BA produced the highest number of shoots (33.70 shoots. Explant -1) and the highest total number of leaves (139.40 leaves. Explant -1), while the longest shoot length achieved with 0.5 mg.l-1 (5.77 cm) as for the kin treatment in the establishment stage, the highest number of shoots (4.50 shoots. Explant -1) and the highest total number of leaves (33.80 leaves. Explant -1) were achieved at 8.0 mg.l-1 , while the longest shoot length (5.02 cm) was achieved at 4.0 mg.l-1. In the multiplication stage, treatment with (2.0 mg.l-1 of kin) produced the highest number of shoots (6.50 shoots. explant -1), the longest shoot length (7.35 cm), and the highest total number of leaves (48.00 leaves. explants -1).