Abstract Proper patient-tailoring strategy and the validation of novel therapeutic targets remain enormous challenges during drug discovery processes. Patient-derived three-dimensional organoid cell culture models possess great potential to associate compound sensitivity and disease complexity in order to provide a key missing link between compound screening and clinical trials. Abemaciclib is a reversible, ATP competitive, selective inhibitor of the kinase activity of both CDK4 and CDK6 and is currently undergoing advanced clinical testing. In this study, we established and characterized three-dimensional organoid cultures from primary colorectal cancer patients and validated their use as drug sensitivity models. We aimed to explore the antitumor activity of abemaciclib in colon cancer organoid cultures by assessing markers for cell viability, proliferation, cell cycle, senescence and apoptosis. Single cell suspension of patient-derived samples were precultured for four days to allow for complete morphogenesis of three-dimensional organoid structures. Subsequently, the cultures were treated for at least two population doubling times and analyzed by luminescent cell viability, immunohistochemistry and flow cytometry assays. Our data suggest that abemaciclib treatment decreased the cell viability of patient-derived colorectal cancer organoid cultures characterized by G1 cell cycle arrest and reduced Ki-67-positive cells. Furthermore, treated cultures showed elevated levels of reactive oxygen species and increased markers for early and late apoptosis. In summary, complex organoid models have the potential to further evaluate the antitumor activity of abemaciclib in various tumor types by enabling mechanistic studies in a patient-specific preclinical setting. Citation Format: Karsten Boehnke, Bruna Calsina, Joaquín Amat, Ana Hermoso, Raquel Torres, Christoph Reinhard, Juan A. Velasco, Philip W. Iversen, Alfonso De Dios, Sean Buchanan, Richard P. Beckmann, Dirk Schumacher, Christian RA Regenbrecht, Marie-Laure Yaspo, Hans Lehrach, María José Lallena. Preclinical analysis and characterization of abemaciclib using three-dimensional patient-derived colorectal cancer organoid cultures. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2829.