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Background/Objectives: Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have a poor prognosis. Local therapy may enhance tumor control and increase resectability. Intratumoral injection of radioactive holmium-166 microspheres presents a promising and minimally invasive treatment with multimodality imaging capabilities (SPECT, CT, MRI). However, holmium-166 microspheres are not commonly used for intratumoral injections, and PDAC is notorious for its high intratumoral pressure. This study developed an intratumoral injection method with nonradioactive holmium-165 microspheres in ex vivo human PDAC specimens using a novel injection system for suspension homogenization. Methods: An injection system was developed and validated in a laboratory setting. Thereafter, intratumoral injections in surgically removed ex vivo PDACs were performed, and parameters were established to optimize feasibility, defined by the ability to inject and control the microsphere distribution. Also, injection limitations and cutoff values were determined. The distribution was assessed by visual confirmation, CT, MRI, ultrasound, and histopathology. Results: With a validated injection system, intratumoral injections were performed in ten ex vivo PDAC samples. Feasible injection guidelines include but are not limited to ultrasound or CT needle guidance, a maximum injection volume of <20.0% from the tumor volume, ≤3 needle positions, and an injection volume of 0.3–1.0 mL per needle position. Conclusions: Intratumoral injection of holmium-165 microspheres in ex vivo pancreatic ductal adenocarcinoma was feasible with adherence to injection parameters necessary for effective intratumoral deposition and minimal leakage. The injection system and parameters developed here provide a foundation for future studies on holmium-166 microsphere injections in pancreatic cancer patients, with the aim to improve local tumor control as a part of a multimodal therapy. Full article