Dehydrocostus lactone (DHCL), a sesquiterpene lactone is well-known for its antiulcer, anti-hepatotoxic and anticancer activity. However, the studies concerning the safety/toxicity potential of DHCL toward the cells of normal origin remain unclear. The present study is aimed at investigating the toxicity potential of DHCL in renal distal tubular and interstitial fibroblast cell lines (MDCK and NRK-49F cells, respectively), and also in ovarian epithelial cell line (CHO cells). The MTT assay has predicted potential cytotoxic activity of DHCL against the cell line types with IC₅₀ values of 0.99, 2.1 and 5.15 μM, respectively. The prominent dose-dependent (IC_{30,50 & 70}) increase in the percentage of cells at subG1 phase in all the cell lines revealed apoptosis induction, further establishing the cytotoxic effect of DHCL. The DHCL exposure (4 h) revealed the induction of ROS in both renal cell lines, which is responsible for apoptosis induction. The NRK-49F cells displayed dose-wise (IC_30-70) increase in chromatin condensation and membranous phosphatidylserine translocation further confirming apoptotic cell death. Also, their increase in BAX/Bcl-2 ratio, mitochondrial membrane permeability and caspase-3/7 activity establishes mitochondrial mediated apoptosis. In case of CHO cells, the higher percentage of cells at G2/M phase and expression of Cyclin B1 at lower concentration of DHCL (≤IC₃₀), indicate mitotic arrest. The incidence of chromatid gaps and negligible micronuclei formation in treated cells (IC_10–30) suggest that sub-lethal concentrations of DHCL exposure causes mitotic arrest in response to the damages by steady expression of Cyclin B1. Under in vitro condition, the study of DHCL’s potential cytotoxic effect on both kidney cells and ovarian epithelial cells indicated the possibility of adverse effects on normal healthy cells as well. Hence, the study recommends in-depth investigations on DHCL usage concerning its safety in therapeutic applications.