Abstract Models that accurately reflect patient drug response are essential for the clinical design of personalized treatment plans and necessary for preclinical drug development. The advancement of predictive models for rare tumor types is impeded in part, by the relative scarcity of fresh tumor tissue available for study. To address the problem of tissue availability we have developed a label-free, combined functional and chemical selection method for the isolation of rare tumor cancer stem cells (CSC) and circulating tumor cells (CTC) from primary patient tissue and blood. Enriched cells were expanded as 3D microtumors under optimized conditions, validated as CSC through in vivo tumorigenesis studies, and characterized by correlative genomic, proteomic/phosphoproteomic, and phenomic analysis. We found isolation and expansion by this method yielded a source of primary cells suitable for live, cell-based predictive drug screening in multiple rare tumor derived models of neuroendocrine and mesenchymal origin, including locally or regionally advanced and metastatic SCLC, recurrent Merkel Cell Carcinoma, recurrent osteosarcoma and dermatofibrosarcoma protuberans. The 3DKUBE™ rare tumor assay was performed using validated CSCs cultured as perfused 3D microtumors (pMTs). Drug studies using the perfused, 3DKUBE™ rare tumor assay modeled individual patient response better than CSC-based or 3D static microtumor-based drug screens and thus demonstrate the effectiveness of this platform for predictive modeling of individual patient drug response. Taken together, this system provides a means of performing ex vivo drug response experiments on very small tissue samples, including core biopsies, with relevant results for patients. Citation Format: Melissa Millard, Kathryn M. Appleton, Ashley Elrod, Nicholas W. Bateman, Tamara Abulez, Kelly Conrads, Brian Hood, Thomas P. Conrads, Lillia M. Holmes, Teresa M. DesRochers. The perfused 3DKUBE™ rare tumor assay models in vivo drug response [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 6018.