Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects upon the accumulation of unrepaired damage to macromolecules, notably DNA. The ability of ionizing radiation to prevent malignant cells from replicating and to cause their demise is indeed an integral component of the anticancer activity of RT. Neoplastic cells are generally more sensitive to the cytostatic and cytotoxic effects of RT than their healthy counterparts as they exhibit increased proliferative rate and limited capacity for DNA repair. This provides a rather comfortable therapeutic window for clinical RT usage, especially with the development of novel, technologically superior RT modalities that minimize the exposure of normal tissues. Thus, while accumulating evidence indicates that cancer control by RT also involves the activation of tumor-targeting immune responses, assessing cell cycle progression in irradiated cells remains a central approach for investigating radiosensitivity in preclinical tumor models. Here, we detail a simple, flow cytometry-assisted method to simultaneously assess cell cycle distribution and active DNA replication in cultured estrogen receptor (ER)+ breast cancer MCF7 cells. With minimal variations, the same technique can be straightforwardly implemented to a large panel of human and mouse cancer cell lines.