Stress is one of the factors that enhance secondary metabolites production in plants. This study reports the effect of altered culture conditions on production of Hispidulin in callus cultures of Millingtonia hortensis. M. hortensis L.f (Bignoniaceae) is an important tree with traditional medicinal value that grows widely in India, Burma, Southern China and Thailand. The tree is a source of Hispidulin, a bioactive flavonoid with several therapeutic properties. Hispidulin was extracted and quantified from leaves of the tree in nature and compared with the amount of the same, produced using in vitro callus. Murashige and Skoog media with combinations of auxins (2,4-Dichlorophenoxy acetic acid and Indole-3-acetic acid) and cytokinins (6-Benzylamino purine and Kinetin) was used for callus production. Combination of Indole-3-acetic acid: Kinetin (5:5 mg/L) resulted in highest amount of callus in a culture period of 6 weeks. Supplementation of 100 g/L of Polyethylene Glycol (PEG) in the culture medium resulted in an increase of Hispidulin production as compared to control in the 5th week. Extension of the culture period by 3 weeks with incubation temperature of 18ᴼC resulted in increased production of Hispidulin. Quantification was done using HPLC technique on weekly basis. A two fold increase in the concentration of Hispidulin was obtained from in vitro calli as compared to natural leaf extract. The present study offers a renewable and sustainable method for production of Hispidulin using callus culture.