Blak henban )Hyoscyamus Niger L.) is one of the important medicinal plants containing alkaloids, which has played a very important role in the pharmaceutical industry. Increasing the production of these secondary metabolites is often done with special compounds called elicitors or stimulants. Chitosan is one of the stimulants involved in the production of secondary metabolites such as alkaloids. Based on this, a factorial experiment was conducted in the form of a completely randomized design with two factors including chitosan at four levels (150, 100, 50, 0 μM) and in three time periods (24, 48 and 72 hours) with three replications. After suspension of callus in different concentrations of chitosan in different time intervals, they were removed from the medium and examined. The studied traits included total alkaloid, total phenol, and total flavonoids, which were measured using a spectrophotometer. Based on the results of analysis of variance, the simple and reciprocal effects of both time and chitosan treatments made the results significant. The highest amount of total alkaloid was obtained in the treatment containing 100 mg/liter chitosan and in 48 hours. The highest amount of phenol was extracted at the rate of 0.074 mg in the concentration of 100 mg of chitosan and in 72 hours. The highest amount of flavonoid was obtained at the rate of 0.095 mg in the treatment containing 100 mg/liter chitosan and in 72 hours. According to these results and previous findings of other researchers, chitosan is an efficient stimulant to increase secondary metabolites in plants.