Plant metabolites have been used in human life for a long time due to their medicinal and economic value. Alkaloids are a very diverse group of plant metabolites classified into different groups based on their biochemical properties. Biosynthesis of these substances is mainly produced in higher plants. The use of hairy root culture results in stability or an increase in the production of a fixed crop under hormone-free culture conditions. Rapid growth, reduced time of root production, ease of storage, and the ability to synthesize a group of chemical compounds by hairy root cultures are additional advantages for producing valuable secondary metabolites. These roots can also synthesize more than a specific metabolite, so they have high economic value. These roots can also synthesize more than a specific metabolite, so they have high economic value. One effective way to increase hairy root production is to use elicitors. Hyoscyamus niger contains the alkaloid tropane, including atropine (hyoscyamine) and scopolamine (hyoscine), widely used in the pharmaceutical industry. Therefore, hairy root production should be done by contamination with Agrobacterium rhizogenes (Agrobacterium rhizogenes) and examined with roots produced through natural culture. Common roots and hairy roots were also treated with jasmonic acid, methyl jasmonate and salicylic acid at different concentrations. The amount of hyoscyamine and hyoscine alkaloids in the treated roots were evaluated using HPLC method. The results showed that the amount of atropine and scopolamine alkaloids increased up to 5 times compared to normal conditions through hair roots treated with elicitors.