<p>Abundant new nanomaterials are developed every year to enhance traditional radiotherapy by improving therapeutic response and reducing side effects. However, current pre-clinical models for evaluating these nanomaterials suffer from low fidelity (e.g., 2D cellular assays) or high cost (e.g., model animals). Considering that tumor spheroids can proliferate on a large scale and at low cost while possessing complex architectures that replicate in vivo solid tumors, in this work, we developed a microfluidic platform integrated with uniform tumor spheroids for the fast and reliable evaluation of radio-therapeutic nanomaterials. Utilizing a cascade of two microfluidic chips (G-chip and T-chip), our platform enabled fast generation, long-term cultivation, and precise pre-treatment of uniform tumor spheroids. The narrow size distribution and high viability of the tumor spheroids originating from the droplet microfluidic G-chip ensured the subsequent evaluation was reproducible and reliable. By regulating the concentration gradient formed in the T-chip through controlling fluidic flow rates, tumor spheroids can be pretreated with different concentrations of radio-therapeutic nanomaterials precisely and simultaneously. Herein, a previously reported radio-therapeutic nanoenzyme was employed to implement radiotherapy evaluation based on the platform as a proof of concept, producing results with high fidelity to in vivo solid tumor. This microfluidic platform shows great potential for the straightforward, reliable, and cost-effective evaluation of radiotherapy nanomaterials.</p>