Plant cell cultures have been shown as feasible systems for the production of secondary metabolites, elicitation with biotic or abiotic stimuli being the the most efficient strategy to increase the production of these metabolites. Glycrrhiza glabra is one of the well known medicinal plants rich in flavonoids. The potential benefits of Glycyrrhiza flavonoids on human health have made it one of the well studied grou of phytochemicals. However, reports on elicitation and gene expression studies of phenylpropanoid pathway enzymes in Glycrrhiza glabra callus cultures are meager. Therefore, the aim of this study was to evaluate the elicitation effect of chitosan on flavonoid production in callus cultures of Glycrrhiza glabra through Gas Chromatography-Mass Spectrometry and on the expression of flavonoid biosynthetic genes through real-time quantitative reverse transcription-polymerase chain reaction. Chitosan significantly induced the production of flavonoids licochalcone, liquirtigenin and Licoisoflavone B in G.glabra callus culture. Moreover, the production of Licoisoflavone was highly induced as compared to other flavonoids. This induction could be correlated with the higher expression of flavonoid biosynthetic gene, Isoflavone synthase. Expression of Chalcone synthase and chalcone isomerase was also induced by chitosan where as the expression of phenyl alanine ammonia lyase was not induced to a significant level. Therefore, it is hypothesized that the observed response to chitosan is the result of an induction or promotion of the phenylpropanoid pathway, mainly at the branch point leading to the activation of isoflavone synthesis and enhanced production of Licoisoflavone B in Glycyrrhiza glabra callus cultures.