Validation of potential therapeutic targets in cancer requires functional live assays that recapitulate the biology, anatomy, and physiology of human tumors. We present a methodology for maintaining mouse and patient tumor samples ex vivo for in vitro drug-screening as well as for the guidance of patient-specific chemotherapies. The harvested tumor biopsy, excised from mice or patients, is integrated into a support tissue that includes extended stroma and vasculature. The methodology is more representative than tissue culture assays, faster than patient-derived xenograft models, easy to implement, amenable to high-throughput assays and does not carry the ethical issues or expense associated with animal studies. Our physiologically relevant model can be successfully used for high-throughput drug screening.