The objective of this study was to assess the impact of various elicitors on the production of podophyllotoxin (PTOX), a potent anticancer agent, in in-vitro cultures of Podophyllum hexandrum, a plant known for its medicinal properties. The study focused on enhancing the production of PTOX in callus suspension cultures through the application of Chitosan, Sodium Chloride (NaCl), Salicylic Acid (C₇H₆O₃), and Sodium Alginate (NaAlg). Among these, Chitosan proved to be the most effective in stimulating PTOX production, followed by NaCl, NaAlg, and Salicylic Acid. The optimal concentrations for elicitation were determined as 50 mg L^-1 for both Chitosan and NaCl, 15 mg L^-1 for Salicylic Acid, and 20 mg L^-1 for NaAlg. The peak yield of PTOX, measured at 619.33 µg/g dry weight, was achieved using Chitosan at a concentration of 150 mg L^-1. However, it was observed that higher concentrations of Chitosan adversely affected the growth of the cultures. Additionally, the accumulation of PTOX was found to be dependent on the culture's growth phase, with the highest levels recorded during the exponential phase. Remarkably, cell suspension cultures treated with NaAlg demonstrated a fivefold increase in PTOX production compared to the untreated control, highlighting the potential of this approach in enhancing PTOX yield. This study establishes that employing elicitors can significantly augment the production of PTOX in P. hexandrum cultures. Chitosan, in particular, emerges as a promising elicitor for the biosynthesis of PTOX in callus suspension cultures of P. hexandrum.