The tulip (Tulipa gesneriana) is an ornamental herbaceous plant. The aim of this study was to evaluate the effects of plant growth regulators on the evolution of colourful callus in this plant and their relevance to the build-up of certain active glycosides, such as 6-tuliposide A, 6-tuliposide B, tulipalin A, and tulipalin B. The growth regulators 2,4-Dichlorophenoxyacetic acid (2,4-D), Benzyladenine (BA), and Thidiazuron (TDZ) were applied at varying doses in four treatments, and the color, texture, and induction rate of the ensuing callus were assessed. The experiment was conducted using a completely randomized design with ten replicates per treatment under controlled in vitro conditions at the Tissue Culture Laboratory, College of Science for Women, University of Baghdad, Iraq, during the winter season of 2024-2025. The results showed a clear variation in the colors of callus with yellow, green, cream, and red colors recorded. The red callus produced from the (2,4-d one + tdz 1) discourse had the ultimate collection of active compounds recording the highest concentrations of 6-tuliposide A (500.10 µg/ml) and 6-tuliposide B (502.50 µg/ml). In contrast, the cream callus produced from the (2,4-D 1.5+BA 1.5) treatment showed the lowest concentrations of Tulipalin A (81.76 µg/mL) and Tulipalin B (126.43 µg/mL), indicating a close relationship between callus color, the level of metabolic activity, and the synthetic pathways involved in the production of secondary products