Patient-derived xenograft (PDX) and organoids derived from PDX (PDXOs) are ideal pre-clinical models, which recapitulate the tumor heterogeneity. We provide the steps to establish PDX and PDXOs from patients with esophageal squamous cell carcinoma (ESCC) and their utility in examining radiotherapy. We describe steps for generating ESCC-PDXs, four cycles of ionizing radiation treatments using X-ray on PDX, and evaluating radiosensitivity of PDXs. We then detail procedures for establishing and characterizing radioresistant organoids derived from PDX. For complete details on the use and execution of this protocol, please refer to Liu et al., 1 Yang et al., 2 and Chen et al. 3 .