Abstract Purpose: Patient-derived organoid (PDO) is emerging tool for drug sensitivity evaluation. Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Therefore, we set up conditions for breast cancer PDOs and establish an in vitro drug evaluation system for future therapeutic approaches. Methods: All specimens were collected from histologically confirmed breast cancer patients after obtaining IRB approval from the National Cancer Center (NCC2017-0146, NCC2020-0299) under patient’s informed consent. Fifty-four patient samples obtained from surgery or biopsy were delivered to the laboratory within 1-2 hours for organoid establishment. All process for cell isolation from human tissues, criteria of successful culture and passaging, quality control (QC), producing genomic and histologic data were adjusted to make standard operating procedure. For long-term cultured PDOs, we tested 30 drugs including kinase inhibitor or standard therapeutic drugs with concentration from 0.001uM to 100uM using 384well plate and the results were analyzed with AUC data with bortezomib as a control. Results: We optimized organoids culture conditions for breast cancer tissues. Total of 54 tissues included disease subtypes as following: hormone positive (n=32), HER2 positive (n=2), triple-negative (n=19), and other (n=1). Nine organoids were successfully long term cultured over 5 passages and 4 were successfully cryopreserved. Clinic characteristics (TNM stages, subtypes, and disease status) showed no significant association with success of organoid establishment. Also, we confirmed that organoids had similar characteristics to the patients tissue through histological data (H&E, ER, PR, HER2, Ki-67, and CK-19). Measurement of AUC values for tested drugs showed variety range which implicates different drug sensitivity of each organoid. Currently, genetic information of long-term cultured organoid is ongoing with comparative analysis of drug sensitivity. Conclusions: We have established breast cancer organoids which reflect molecular characteristics of the original tumor. This model will serve as the system that can recapitulate drug response profiles of human cancer, and pave the way for screening innovative drugs, identifying novel targets, and stratifying patients for pertinent therapeutic options. (This work was supported by National Cancer Center, Korea (No.2010120) and National Research Foundation of Korea grant, funded by the Korea government (MSIT) (No. 2020M3A9A5036362)) Citation Format: Hyunjin Kim, So-Youn Jung, Sung Hoon Sim, Hee Jung Chae, Yun-Hee Kim, Yena Kim, Eun Gyeoung Lee, Seeyoun Lee, Jai Hong Han, Young Mi Kwon, Eun Sook Lee, Keun Seok Lee, Sun-Young Kong. Target therapeutics evaluation using patient-derived organoids from breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6038.