Advances in biotechnology, particularly methods for culturing and micropropagation of medicinal plants will provide new means for the production of commercial chemicals [1]. Alkaloids represent a highly diverse group of compounds which many of them have been exploited as pharmaceuticals, stimulants, narcotics, and poisons. The scopolamine and its precursor hyoscyamine are found mainly in Solanaceae family and used as parasympatholytics that competitively antagonize acetylcholine [2]. Datura stramonium is a rich source of tropane alkaloids such as atropine, hyoscyamine and scopolamine. In this investigation Datura seeds were cultured on Moorashing and Skoog (MS) medium supplemented with different concentrations of yeast extract (0, 1.5 and 3 g/L). After two month seedlings were gathered and their tropane alkaloids extracted according to the Kamada and et al protocol. The extracts were analyzed by High pressure liquid chromatography (HPLC) for the determination of hyoscyamine and scopolamine contents. The mobile phase was water: acetonitrile (75:25) and flow rate 1 ml/min. The peaks were detected at 215 nmand hyoscyamine sulphate and Scopolamine hydrobromide used as standards [3]. Based on the HPLC chromatograms, scopolamine content of roots in seedlings treated with 1.5 g/L yeast extract was higher than other groups, but in shoots its amount was more or less similar to untreated seedlings. Moreover hyoscyamine content of shoot and root parts of treated with 1.5 g/L yeast extract was higher than the other groups. In conclusion, yeast extract (1.5 g/L) can be used as good stimulator for production of valuable medicinal compounds; hyoscyamine and scopolamine.