Hyoscyamus is one of the important genus in Solanaceae which distribution of some of its species is restricted to Iran and some of the neighboring countries. The main medicinal substances in this plant are alkaloids (tropane alkaloids). One of Hyoscyamus species which has not been analysed yet, is Hyoscyamus insanus.This plant was collected from southern part of Iran and dried in shaddow and after milling was macerated in 96% ethanol. After maceration, total alkaloids were extracted and using successive column and preparative thin layer chomatography, extract fractionated into three parts. The alkaloids is each fraction was identified using Nuclear Magnetic Resonance (NMR) spectroscopy, Mass Spectrometry (MS) and Ultra Violet (UV) spectrophotometry. These three alkaloids were hyoscyamine (atropine), hyoscine and apoatropin. More than 70% of the total alkaloids was hyoscyamine. Regarding this high percentage of hyoscyamin, this species could potentially be used for large scale production. It should be metioned that this is the first report on alkaloid contents of this plant.