Sesame (Sesamum indicum L.) is not only a source of manganese and copper, but is also rich in calcium, magnesium, iron, phosphorus, vitamin B1, zinc and dietary fiber. Sesame contains a group of fibers called Lignans, including Sesamin and Sesamolin, which are especially useful as they decrease cholesterol in the human body. Due to its use in medical treatments, especially in cancer cases, recent decades have seen numerous efforts aimed at a better understanding of Sesamin production and the genes underlying its biosynthesis, with the aim of enhancing expression of genes involved in this pathway. In this study, the effects of salicylic acid and phenylalanine on the expression level of two key genes, CYP81Q1 and C3H, involved in the Sesamin biosynthesis pathway were evaluated using sesame cell suspension culture and qRT-PCR. For this purpose, sesame cell suspensions were treated using 0.1, 0.5 and 1 mg/l salicylic acid and 0.1 mg/l phenylalanine. Sampling was carried out 24, 48 and 72 hours after treatment. Gene expression analysis was performed by the qRT-PCR technique. The results showed that expression of genes involved in Sesamin biosynthesis increased in all the samples treated with salicylic acid and phenylalanine. The highest increase in expression level of CYP81Q1 occurred in 1 mg/l of salicylic acid 72 h and 0.1 mg/l of phenylalanine 72 h after treatment. The highest increase in expression level of the C3H gene occurred in 0.1 mg/l of salicylic acid 24 h and 0.1 mg/l of phenylalanine 72 h after treatment.