The abiotic stress such as water, temperature, and drought influences the production of pharmacologically important secondary metabolites of plant as a defense mechanism. Cassia angustifolia Vahl. is a potent medicinal plant which yields sennosides from its leaves and pods and grow well under water stress condition. Its leaves and pods are used for the treatment of habitual constipation as a safe herbal laxative. The present study aimed to determine the response of Cassia angustifolia Vahl. to water stress in terms of expression of targeted genes involved in the synthesis of its major secondary metabolites i.e., sennosides. The four moisture regimes i.e., 60% available soil moisture (ASM), 40% ASM, 20% ASM, and rainfed along with control were imposed from 20 days after seed sowing to the harvesting of the crop. The four ages of the plant (60 days, 75 days, 90 days, and 105 days) were taken for evaluation of differential gene expression due to water stress. Results demonstrated that water stress significantly affected the morphophysiological characters, sennoside content and gene expression. Sennosides determination was done by performing HPLC, which showed an increasing trend with increasing moisture stress. Proline, MDA, carotenoid, and electrolyte leakage were also increased in support of sennoside increment at higher moisture stress conditions. The gene expression, stress enzymes, and morphophysiological parameters such as chlorophyll, carotenoid, and Fv/Fm demonstrated a significant correlation of sennoside content. The RT-PCR analysis showed the upregulation of genes associated with the Mevalonate (HMGR, PMK), Shikimate (SDH, SMK, CS), Menaquinone (menF), and Glycosylation (UDPG) pathways at rainfed and 20% ASM conditions, suggesting their involvement in increased secondary metabolite production under moisture stress conditions. However, plant age does not have any significant effect on gene expression.