Locally advanced adenocarcinomas located in the distal rectum are commonly treated via 5-fluorouracil (5-FU)-based neoadjuvant chemoradiation therapy (CRT). The occurrence of pre-operative pathological complete response, or the absence of any histological evidence of residual cancer, is seen in 15-27% of rectal cancer cases. Response to chemotherapeutic agents varies between patients, introducing the need for a system to predict optimal drug combinations. We propose a method of utilizing optical metabolic imaging of in vitro, primary tumor-derived, three-dimensional organoid culture to create specific drug sensitivity profiles, and to rapidly assess a patient’s potential response to drugs. Murine xenografts were developed in Swiss athymic nude mice, using human colorectal adenocarcinoma cell lines, implanted in the flank (RKO, ATCC). Tumors were excised upon reaching a volume of 500mm3 and processed for organoid culture. Organoids were subjected to longitudinal metabolic imaging of metabolic cofactors FAD and NADH for seven days. The resulting images were used to yield an optical redox value on a cell-by-cell basis, determined by the fluorescence intensity ratio of FAD/(FAD+NADH). This data infers proliferative index of the organoids. Beginning on day three, a control vehicle dimethyl sulfoxide, or the cytotoxic agent 5-FU, was added to the organoid growth media in wells, with metabolic imaging performed the same as previously stated. The optical redox values decreased due to the addition of 5-FU, which targets rapidly dividing cells and induces apoptosis. The changes in the optical redox histograms were correlated to markers of cell proliferation (Ki-67) and apoptosis (cleaved caspase-3).