An efficient shoot regeneration protocol based on callus induction and multiple shoot development has been standardized for Plectranthus barbatus. The leaf explants were cultured on Murashige and Skoog (MS)  medium supplemented with α-naphthalene acetic acid (NAA at 2.68 - 16.11 μM) alone or in combination with Kinetin (KIN) and 6-benzyladenine (BA) for callus induction. The best callus production was obtained with medium containing NAA 10.74 μM with BA 2.66 μM. After two weeks of growth calli were transferred to shooting medium containing BA 11.10 μM and NAA 3.22 μM, shoot regenerated with a frequency of approximately 28.21 ± 3.10 shoots per callus and maximum average shoot length (8.37 ± 2.03 cm) were recorded on half strength MS medium supplemented with 1.73 μM Gibberellic acid (GA3) and 2.32 μM KIN. Rooting was best achieved on half-strength MS medium augmented with 7.38 μM Indole-3-butyric acid (IBA). The plantlets regen-erated in vitro with well developed shoot and roots were successfully established in pots containing garden soil and grown in a greenhouse with 90% survival rate. The regenerated plants did not show any immediate detectable phenotypic variation. The described method can be successfully employed for large-scale multiplication and long term in vitro conservation of Plectranthus barbatus.