Recently drug‐induced senescence has gained importance in cancer therapy. It has been demonstrated that cardiac glycosides induce senescence in several cancer cell lines. On the other hand, small G protein Rho is involved in the formation of morphological features of the senescent cells. However, there is no report whether Rho kinases, important effectors of Rho, contribute to the development of the senescent morphology in cancer cell lines. In the current work, we evaluated this possibility in the cardiac glycoside‐induced senescence in HeLa cells. The xCELLigence real time cell analyzer system, senescence associated β‐galactosidase staining and cell size measurements were used to determine ouabain‐induced senescence. Cells were treated with high (0.1–1 μM, 48h) and low concentration (10–30 nM, 48h) of ouabain. In contrast to the treatment of ouabain in high concentrations which resulted in cell death, low nanomolar concentrations of ouabain induced senescence in HeLa cells. Although preincubation of specific Rock inhibitor Y‐27632 (0.1–10 μM, 30 min) did not inhibit the occurrence of ouabain‐induced (30 nM) senescence in cells, it reduced the cell size. Our results indicate that instead of the induction of senescence, rho kinases involves in the development of drug‐induced senescence morphology.This study was supported by TUBITAK in 110S426 project.