Abstract BACKGROUND As the leading disease-related cause of death in children, pediatric brain tumors, and in particular diffuse midline glioma, require novel treatment approaches. Cellular-based therapies offer promise, but challenges still exist in defining their optimal delivery route. Emerging data from our group suggest there may be a role for intratumoral injection in treatment algorithms. We here describe a surgical workflow for intratumoral injection of cellular therapies that utilizes existing and widely used neurosurgical technologies that can be incorporated into future clinical trials. METHODS Infusion target and catheter trajectory was planned using standard pre-operative MRI. Components from an FDA-approved frameless stereotactic intracranial biopsy system were chosen such that they would accept and guide an FDA-approved rigid intracranial catheter system intended for MR-guided procedures. Following catheter insertion, an intraoperative CT was used to determine accuracy of catheter placement by merging the image with the pre-operative MRI. RESULTS The proposed system was tested in a simulated fashion using a phantom cranial model registered to an accompanying brain MRI. The stereotactic biopsy mount was affixed to the skull at the planned entry site for a posterior fossa approach to the pons. Catheter insertion depth was determined using the stereotactic system and the catheter was inserted to this depth through the biopsy mount. Intraoperative CT confirmed the catheter tip location which correlated with the planned infusion target with sub-millimetric accuracy. CONCLUSION Our proposed intratumoral delivery method using frameless stereotaxy shows promise for use in future clinical trials that require intratumoral delivery of cellular therapies. Utilizing FDA-approved devices allows for quick integration into protocols and their wide-spread use makes the approach generalizable. This approach also eliminates the need for MR-guided procedures which can increase cost and require significant hospital resources to perform. Future work will be aimed at streamlining the intraoperative workflow.