Purpose: Sustained cell proliferation and cell cycle acceleration in cancer cells inherently increase DNA damage, which interferes with homeostatic replication and transcription. Ataxia telangiectasia and Rad3-related (ATR) is crucial for initiation of the DNA damage response, and ATR inhibitors, such as elimusertib, induce increased replication stress and DNA damage. We investigated the anti-tumor effects of elimusertib and its mechanism of action in relation to replication stress. Materials and Methods: Anti-tumor effects were evaluated by MTT assay and colony formation assay in breast cancer cell lines in vitro, in breast cancer cell xenografts in vivo, and in patient-derived xenograft models. Cell cycle was assessed by flow cytometry and BrdU assay was used to measure replicating cells and S-phase progression. Alkaline and neutral comet assay was used to measure single and double-stranded DNA damages, respectively. Results: Elimusertib delayed S-phase progression in MDA-MB-453 and MDA-MB-231 cells and induced caspase-7–dependent apoptosis. Furthermore, the increase in sub-G1 population in the fluorescence-activated cell sorting analysis and Annexin V assay also confirmed apoptotic cell death. In the BrdU assay, single-stranded DNA (ssDNA) increased in sensitive cells and aberrant ssDNA induced DNA damage in S-phase and eventually caused replication catastrophe. Finally, these anti-tumor effects were proven in in vivo xenograft and patient-derived xenograft models. Conclusion Elimusertib had anti-tumor effects and induced replication catastrophe in breast cancer cells with a high replication rate. Moreover, cells under high DNA replication stress were sensitive to elimusertib. Further studies and treatment strategies with elimusertib are warranted for cancers with a high replication rate.