<p>Tumor heterogeneity drives chemotherapy resistance in cancer, necessitating the development of strategies to target diverse cellular subpopulations. Single-cell-derived tumor organoids (STOs), derived from individual tumor stem cells (TSCs), retain well-defined genomic and phenotypic attributes, making them instrumental in elucidating unique diversification. Here, we developed a microfluidic approach that utilizes STO arrays for personalized drug screening, aimed at overcoming heterogeneity induced drug resistance in colorectal cancer. We utilized a microwell array format microfluidic chip to the generation of a high scale STO array. This array encompasses two key features: (1) STOs in individual microwells are derived from single TSCs, which allows for a high-resolution display of cellular heterogeneity; (2) it exhibits thousands of STOs at predefined locations on the same focal plane, thereby facilitating automated, high-content analyses using imaging data. Utilizing the STO array, we assess the responses of various subpopulations of colorectal cancer to a conventional chemotherapeutic regimen, facilitating the phenotypic identification of resistant STOs. Through the analysis of RNA sequencing data from the resistant STOs, candidate drugs targeting resistance-associated pathways were identified for individual patients. Furthermore, the candidate drugs were validated through STO-array drug testing, demonstrating enhanced clearance effects when combined with the conventional regimen. Our study establishes a scalable, patient-specific drug screening approach that integrates phenotypic and molecular analyses, offering a transformative strategy to overcome heterogeneity-driven drug resistance in colorectal cancer. The microfluidic platform presented herein holds significant potential for advancing precision oncology and personalized therapeutic design.</p><p></p>