The lack of progress in treatment advances for glioblastoma (GBM) over the past decade underlines the necessity of deepening our molecular understanding of how glioma cells survive chemoradiation and instigate recurrence1,2. Quiescent glioma stem cells (GSCs) have been identified as a key factor of treatment resistance, however, progress in targeting and understanding these cells has been hampered due to challenges such as ambiguous cell markers and dedifferentiation of glioma cells into stem-like states3,4,5. To overcome this issue, an in vivo model of GBM, which allows for the direct visualisation and tracking of quiescent GSCs is required. Here we established a novel, orthotopic quiescence tracking mouse model (QuiT model) using somatic-cell electroporation of plasmids. These plasmids combine Cas9-knockout of GBM-oncogenes Nf1, Pten and Trp53 (NPP)6 with the labelling of quiescent GSCs through a kinase-dead mVenus-P27 construct downstream of a Prom1 promoter7. We further modified the NPP plasmid by replacing the cytoplasmic fluorescent reporter with a nuclear membrane-bound fluorescent reporter to facilitate single-nucleus RNA-sequencing studies. Additionally, we optimised plasmid-transposition efficiency to enable over-expression or knockout studies via the addition of a third, doxycycline-inducible plasmid. We propose the QuiT model as a fast and biologically relevant tool to determine the best therapeutic strategies to target these quiescent GSCs.