Diffuse midline glioma (DMG) is a highly aggressive brain tumor that predominantly affects children. Conventional treatments such as radiation therapy can control progression for a time, but DMG kills nearly 100 percent of patients. Although murine models have provided critical insights into the biology of DMG, they are not suitable for high-throughput screening to identify and profile novel therapies due to technical challenges, ethical considerations, and high cost. Zebrafish (Danio rerio) is an established vertebrate model for large-scale drug screening, and zebrafish have demonstrated the ability to replicate the key biological and pathological aspects of human malignancies. Here, we developed a novel method for transplanting human DMG cells into large numbers of zebrafish embryos to speed the assessment of anti-tumor drug efficacy in vivo and thereby facilitate the development of novel therapeutics for clinical translation. We transplanted red fluorescent protein (RFP)-labeled, patient-derived DMG cell lines into zebrafish blastulas. Remarkably, many DMG cells migrate into the developing brain and are present in the midline of the brain 24 h after blastula injection. Tumor cell burden was monitored by measuring RFP fluorescence intensity changes over time. Time-course images of transplanted tumor cell volumes were acquired, and the interactions between transplanted DMG cells and microglial cells were further analyzed using Imaris software. We have developed a robust and rapid transplantation protocol to establish a zebrafish xenograft model of DMG. Unlike existing orthotopic zebrafish models, our protocol eliminates the need for technically demanding intracranial transplantation into individual embryos. This approach allows for the transplantation of hundreds of embryos per hour, thereby offering a scalable and orthotopic vertebrate DMG model that is directly applicable to high-throughput drug and gene discovery screens, which have the potential to accelerate clinical translation.