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Patient-derived organotypic tumor spheroids (PDOTS) are short-term, 3D cultures of multicellular spheroids in microfluidic devices, prepared from freshly explanted patient tumors. In this protocol, we describe the steps of PDOTS sample processing and culturing, drug sensitivity testing ex vivo using a microscopy-based assay, and the assembly of a PDOTS cohort that captures the overall response with patient-to-patient diversity. We highlight critical technical steps and call attention to potential pitfalls, offering troubleshooting guidance and important quality control details. For complete details on the use and execution of this protocol, please refer to Revach et al..