Withania somnifera is interest plant. It’s a distinguished medicinal plants in Indian systems of medicine, and many other places in the world this important plant used against many type of diseases, due to a many of different classes of chemical constituents that contains in Ashwagandha such as: steroidal lactones, alkaloids, flavonoids, saponine, somniferine and Iron. Due to these important chemical constituent Ashwagandha have different pharmacological activity such as anti-inflammatory, anticancer, antidiabetic, ant stress, antimicrobial, neuroprotective, cardio protective, modulate mitochondrial activity and reinforce endothelial function. The plant used single or with other medicine plants as combination to treat many type of humane diseases. Phytochemical screening of Iraqi Withania somnifera with the isolation and quantitative identification of some secondary metabolites. Using the root of plant, the extraction stage was preceded by a defatting step using n-hexane for 48 hours , followed by typical technique of extraction that were used in this work for plant material, Cold technique by maceration with 85 percent methanol followed by further fractionation by using different solvent according to increase of polarity, petroleum ether, chloroform, ethyl acetate, n-butanol and separation using high-performance liquid chromatography (HPLC),the Hexane, chloroform fractions was analyzed by thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and the identification of isolated compounds by using ultraviolet (UV), high-performance thin layer chromatography (HPTLC), thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) gas chromatography-mass spectrometry (GC-MS) was used for further analysis for the hexane fractions. Preliminary chemical testing revealed the presence of alkaloids, steroids, terpenoids, in the extracted substance. The separation of three phytoconstituents found by various chromatographic methods resulted in the confirmation of the non-polar chemicals separated from the hexane fraction, which included the phytosterol: Stigmasterol, Beta sitosterol with Caffeine was separated from the chloroform fraction. The secondary metabolites contained in the Iraqi Withania somnifera plant have a wide range of therapeutic effects. Caffeine will be the first isolation from the Withania plant, and beta sitosterol and stigmasterol is also the first isolation from the species near Iraq.