The present study attempts to explain a mass culture protocol for large scale multiple shoot bud regeneration from nodal explants of Citrullus colocynthis L. The highest shoot bud induction (94.9%) without attaining callus was observed in Murashige and Skoog’s (MS) medium supplemented with benzyl amino purine (BA)in which the combination of benzyl amino purine BAP (1.0 mg/l), kinetin (0.5 mg/l), gibberellic acid (1.5 mg/l) produced more shoots/explants. This study strongly suggests that the pre-eminence of BA and other cytokinins in combination with GA3 was found to be essential for a swift multiple shoot bud induction as well as an enhanced rate of shoot proliferation in C. colocynthis. A full-strength MS medium, supplemented with indole 3 butyric acid IBA (1.5 mg/l) combined with BA (1.5 mg/l), had the maximum rooting response and the regenerated plantlets were successfully established in the field with an 80% survival rate.