Context: Psoralea corylifolia L. (Fabacese) is rich source of bioactive compounds, which endows the plant with immense value for its use in pharmaceuticals, health, and body-care products. Objective: The current study was designed (i) for the determination of psoralen from callus derived from different plant parts, and (ii) for the enhancement of psoralen in in vitro condition with the treatment of various psoralen pathway precursors. Materials and methods: B5 media were employed for raising the cultures from different plant parts such as leaf, node, root, and green seeds. Cotyledons’ calluses were derived from cotyledon of green seeds that were elicited on MS + 10 mM BA+5mM IBA medium supplemented at 0.1, 1, 2.5, 5, 25, and 50 mg/L of various psoralen pathway precursors such as umbelliferone, cinnamic acid, and NADPH. The method for extraction of psoralen was modified from the Singh method and the content of psoralen was measured using HPLC. Results: HPLC analysis of callus derived from different parts of P. corylifolia revealed that a maximum of psoralen (2601.8 mg/g fresh wt.) was recorded in cotyledons’ callus. Cotyledonary callus was chosen for the enhancement of psoralens because of higher amount of psoralen in it. In vitro evaluation showed that all the precursors enhanced the psoralen amount dramatically so that the optimum amount of psoralen (2518.8 mg/g fresh wt.) was obtained at 2.5 mg/L cinnamic acid. Discussion and conclusion: The results obtained indicate that cinnamic acid is one of the important precursors of psoralen pathway that induced a maximum amount of psoralen with in vitro conditions.