Abstract Background: The I-SPY 2 TRIAL is a multi-site response adaptive clinical trial evaluating novel drug combinations for neoadjuvant treatment of breast cancer. Patients receive four or more MRI studies during treatment, and serial measurement of functional tumor volume (FTV) is used to assess response. Under FDA IDE approval, FTV plays an integral role in adjusting patient randomization and evaluating treatment efficacy. FTV is a quantitative measure that requires stringent standards for image quality and protocol adherence. The I-SPY 2 TRIAL consistently reports a high level of data quality and data acceptance for FTV measurements. We present an overview of MRI operational performance and share lessons learned about maintaining high quality MRI data in a multi-site clinical trial. Methods: Over the 10-year course of the I-SPY 2 TRIAL, workflow has been improved to optimize communication between the Imaging Core Lab (ICL) and sites and to collect details about the MRI that are needed for accurate FTV measurement. A standardized imaging acquisition protocol is distributed to all sites, and new sites submit two test cases for review at site initiation. A scan verification form is required for each MRI study completed at sites to document critical information. Sites submit studies using TRIAD image transfer and de-identification software (American College of Radiology), and data is archived and processed at the ICL. All MRI studies are reviewed by the ICL for protocol adherence as soon as they are submitted, and feedback is provided to sites. Image quality factors including motion, fat suppression, and signal-to-noise ratio are qualitatively assessed. The ICL communicates with sites through a centralized email account, regular Coordinator Calls, and Imaging Working Group meetings to discuss emerging issues and offer ongoing training. The ICL contributes to revisions of study protocols and standard operating procedures. Results: As of June 2020, 3020 patients had been registered in I-SPY 2, 1741 patients randomized to treatment with one of 18 experimental drugs or standard therapy (controls), and a total of 7527 MRI studies were performed. FTV could be calculated for 97% (7317/7527) of MRI studies. Of the 7317 studies where FTV could be calculated, relatively minor issues with image quality or imaging protocol adherence were documented for 28% (2030/7317). These issues included motion artifacts (32%, 659/2030), off-protocol scan duration (21%, 433/2030), off-protocol contrast injection rate (14%, 281/2030), and off-protocol imaging field of view (9%, 191/2030). Conclusion: Breast MRI studies using a variety of scan protocols are well-suited for diagnostic evaluation, including BIRADS categorization, measurement of longest diameter, and assessment of lesion washout. The quantitative measures used in the I-SPY 2 trial require adherence to a specific imaging protocol that is kept consistent for all MRI studies for a single patient. Operational standardization, clear communication with sites, and streamlined workflow yield high quality MRI data across multiple sites and scanners. As a result, FTV is a robust biomarker of response to treatment, and is being used to predict patient response and guide treatment planning. We are actively investigating strategies that will improve FTV accuracy for predicting response and informing guidelines for treatment de-escalation. This will allow the ICL to further standardize and improve image quality and will provide the foundation for testing a variety of imaging biomarkers in the I-SPY 2 TRIAL. Citation Format: Jessica Gibbs, David C Newitt, Margarita Watkins, Wen Li, Lisa Cimino, Clifton Li, Natsuko Onishi, Lisa J Wilmes, Teffany Joy Bareng, Evelyn Proctor, Barbara LeStage, Bev Parker, the I-SPY 2 Coodinators, the I-SPY 2 Imaging Working Group, Nola M Hylton. Operational standardization and quality assurance yield high acceptance rate for breast MRI in the I-SPY 2 TRIAL [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-08.