Abstract: Tissue culture has been established as a useful ex situ conservation strategy for Aloe species. This paper describes in vitro regeneration of Aloe barbadensis (Liliaceae) and preliminary estimation of secondary metabolites in methanolic extract of in vitro regenerated plants. Shoot tips were used as explants for in vitro regeneration on MS medium supplemented with 4 mg L-1 Benzyl amono purine (BA) and 1 mg L-1 Indole acetic acid (IAA). In Vitro rooting was induced when Murashige and Skoog’s (MS) medium was supplemented with 1 mg L-1 Indole Butyric Acid (IBA). Thin layer chromatography studies were carried out on methanolic extract of in vitro regenerated plants.