Patient-derived tumor organoids (PDTOs) have the potential to be used to predict the patient response to chemotherapy. However, the cutoff value of the half-maximal inhibition concentration (IC50) for PDTO drug sensitivity has not been validated with clinical cohort data. We established PDTOs and performed a drug test in 277 samples from 242 CRC patients who received FOLFOX or XELOX chemotherapy. After follow-up and comparison of the PDTO drug test and final clinical outcome results, the optimal IC50 cutoff value for PDTO drug sensitivity was 43.26 mmol/L. This PDTO drug test-defined cutoff value could predict patient response with 75.36% sensitivity, 74.68% specificity, and 75% accuracy. Moreover, this value distinguished groups of patients with significant differences in survival benefit. Our study is the first to define the IC50 cutoff value for the PDTO drug test to effectively distinguish CRC patients with chemosensitivity or nonsensitivity and predict survival benefits.