The present study focused on optimizing callus induction and multiple shoot cultures of Psoralea corylifolia Linn. by testing various combinations and concentrations of plant growth regulators (PGRs). The highest callus induction was achieved using leaf explants, followed by stem and root explants. Optimal callus induction was observed with 2.0 mg/L of 2,4-D + NAA and 3.0 mg/L of IAA in Murashige and Skoog's medium, which resulted in distinct embryogenic callus formation. Multiple shoot cultures were successfully induced on MS medium supplemented with 2.0 mg/L 2,4-D + BAP, producing an average of (15.9 ± 0.4) shoots per explant. The study also investigated the effects of salicylic acid (SA) elicitation on biomass and psoralen content. SA showed dose-dependent effects, with the 10 µM concentration leading to a 2.11-fold increase in fresh weight and the highest psoralen content of 5.6% dry weight observed at 20 µM. Additionally, 4.3% dry weight of psoralen was observed at 10 µM within the first week compared to controls. This research highlights the importance of optimizing PGR concentrations for efficient multiple-shoot induction via callus and the use of SA elicitation to enhance secondary metabolite production in P. corylifolia. These findings have significant implications for the rapid multiplication of plant material for conservation and commercial purposes. Future efforts should focus on scaling up these findings for practical application in conservation and biotechnology