ABSTRACT Aloe vera (L.) Burm. f., Xanthorrhoeaceae, a succulent, produces barbaloin, a bioactive compounds used in various pharmaceutical products. Extracts prepared from the leaves have been widely used as bittering agents, taste modifiers and also as cathartic agent against severe constipation. Barbaloin is reported for its anti-inflammatory, anticancer, antiviral and anticancer activities and these properties are mostly mediated by its antioxidative capacity. Presently, a study has been conducted on the comparative High Performance Thin Layer Chromatography analysis of barbaloin from the dried leaf skin powder of in vivo and in vitro grown A. vera. Shoot tips of A. vera were cultured in Murashige and Skoog media supplemented with different combination of 6-benzylaminopurine and 1-naphthaleneacetic acid. [Best multiplication response was noted in benzylaminopurine (2.0 mg/l) + 1-naphthaleneacetic acid (0.1 mg/l) supplemented Murashige and Skoog media]. The quantitative determination of barbaloin was performed on silica gel 60 F254 HPTLC plates as stationary phase. The linear ascending development was carried out in a twin trough glass chamber saturated with a mobile phase consisting of ethyl acetate: methanol: water (100:16.5:13.5) at room temperature (22 ± 2 ºC). CAMAG Thin Layer Chromatography scanner-3 equipped with CATS software (version: 1.4.4.6337) was used for spectrodensitometric scanning and analysis in the ultraviolet region at λ = 366 nm. The method was validated for linearity, precision and accuracy. Correlation coefficient, limit of detection, limit of quantification as well as recovery values were found to be satisfactory. Out of the five populations studied, the leaf skin of A. vera collected from Jodhpur (Rajasthan, India) and raised in vitro was found to contain higher amount of barbaloin (2.78%) when compared to its naturally growing counterparts (2.46%) and other plant populations.