This study was conducted to establish an efficient in vitro plantlet regeneration protocol using the ex vitro leaves as explants for Haworthia retusa. Leaf tissues were cultured on liquid full-strength Murashige and Skoog (MS) medium supplemented with 2.0 mg/L indole 3-butyric acids (IBA) for callus induction, followed by sub-cultured to solid medium for callus proliferation. Callus was then transferred to a fresh medium supplemented with 6-benzyl amino adenine (BA) for shoot development. The result showed that the maximum rate of shoot regeneration (100%), number of shoots per explant (43), and shoot height (9.4 mm) were recorded on the solid MS medium supplemented with 1.0 mg/L BA and 30 g/L sucrose. IBA improved rooting, whereas, NAA (naphthaleneacetic acid) causes calli to form at the base of the shoots. The half-strength MS medium supplemented with 0.5 mg/L IBA provided the best rooting response for the shoot. This medium formulation resulted in the highest rooting rate (100%) and the highest mean root number (5 roots/explant). The result of the present study would be helpful for the mass propagation of commercially important H. retusa.