Medicago sativa L. (alfalfa) is one of the most cultivated plants which has high nutritional value as fodder since antiquity and to treat many health disorders related to secondary metabolites with antioxidant and anti-inflammatory effects. For the first time, a comparative study was applied for bioactive constituents of tissue cultured and their ordinary counterparts of Iraqi Medicago sativa. Two types of agriculture techniques were employed, plant tissue cultured (three different tissue culture types) and ordinary agriculture (two different aerial parts and growth stages). The reverse - phase high-performance liquid chromatography applied for qualitative and quantitative of different phytochemicals in this plant giving an idea about the differences in the availabilities of these phytochemicals like steroids (Beta-sitosterol, Stigmasterol), phenolic acids(Caffeic acid, Chlorogenic acid, Gallic acid), and flavonoids(Myricetine, Naringinine) by the differentiated techniques.