Abstract Radiotherapy (RT) plays a crucial role in the treatment of cervical cancer. However, the efficacy of existing radiosensitizers in clinical applications is limited. The aim of this study was to established an efficient method of identifying new radiosensitizers and to investigated the specific mechanisms when used in combination with RT. An effective platform for radiosensitizers discovery was created based on high-throughput screening (HTS) technology. We established an automated and powerful HTS approach to identify potent radiosensitizers from the LOPAC1280 chemical library. We used the Geldanamycin for validation of our HTS method, which had been known as a radiosensitizer for cervical cancer cells. Z'-score of our HTS platform was 0.6. From 1280 compounds, we identified four hit compounds. Among them, we found Dihydroouabain (DHO) and Ro90-7501 which have not been previously known as radiosensitizers. Clonogenic survival assay showed that DHO and Ro90-7501 significantly enhanced radiosensitivity in cervical cancer cells. Western blot analysis showed that DHO inhibited the phosphorylation of Chk1, and Ro90-7501 inhibited the phosphorylation of ATM. These results indicated that DHO and Ro90-7501 inhibited DNA damage response pathway. In conclusion, we established the HTS method for radiosensitizer screening, and found DHO and Ro90-7501 as new radiosensitizers targeting DNA damage repair pathway. Citation Format: Keisuke Tamari, Zhihao Li, Hideki Matsutani, Yuji Seo, Yutaka Takahashi, Kazumasa Minami, Fumiaki Isohashi, Kazuhiko Ogawa. High throughput screening identified new radiosensitizers which inhibit DNA damage repair pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6171.