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Background Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor in adults. Myeloid-derived suppressor cells (MDSCs), particularly the granulocytic subset (gMDSCs), are highly abundant in GBM and suppress anti-tumor immunity, making them a high-priority therapeutic target. In mice, gMDSCs are identified by Ly6G expression. Due to tissue limitations, tumor heterogeneity, and the need to assess gMDSC levels in survival studies without euthanasia, we aimed to develop a noninvasive tool to quantify gMDSCs in preclinical GBM using positron emission tomography (PET).Methods Flow cytometry confirmed Ly6G+ gMDSC infiltration in orthotopic syngeneic GBM tumors. An anti-Ly6G antibody (clone 1A8) was conjugated to a DFO chelator and radiolabeled with Zirconium-89 ( 89Zr). Longitudinal PET/CT imaging and biodistribution studies were performed at 48- and 120-hours post-injection in tumor-bearing mice injected with resulting radio-conjugate, 89Zr-DFO-anti-Ly6G, at multiple specific activities as compared to blocking and vector controls.Results Our data confirms high MDSC levels in murine GBM. The radiolabeled tracer ( 89Zr-DFO-anti-Ly6G) was produced with high radiolabeling yield (>95%) and in vivo stability over 120 hours. Initial PET imaging demonstrated that tumor uptake increased at 100 µg (SUVmean = 3.08 ± 0.101) vs. 40 µg (SUVmean = 1.98 ± 1.062). Additionally, pre-injection of cold antibody (25x) enhanced tumor uptake by blocking peripheral Ly6G, reducing tracer accumulation in blood and immune organs while increasing tumor signal (SUVmean = 3.25 ± 0.724).Isotype controls showed a strong correlation between tumor and non-tumor uptake (SUVmean = 1.59 ± 0.703), indicating peripheral receptor saturation as a mechanism of enhanced tumor targeting. Tumor tracer uptake increased over time in all groups. Ex vivo biodistribution studies confirmed PET imaging findings. PET imaging studies are currently underway to further optimize specific activity identify an optimal balance between peripheral blocking and tumor targeting, and further validate specificity of 89Zr-DFO-anti-Ly6G through blocking (100x) studies.Conclusions gMDSCs are a critical therapeutic target in GBM. We developed Ly6G-PET, a novel, non-invasive imaging strategy using 89Zr-DFO-anti-Ly6G to detect and monitor gMDSCs in vivo. This approach enables longitudinal tracking of gMDSCs in preclinical models and supports the clinical potential of MDSC-targeted PET imaging in GBM.