Background Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite the standard of care treatment of surgery, radiation therapy and Temozolomide (TMZ) chemotherapy, GBM frequently recurs in a drug-resistant form. This work investigates how inhibition of the hyperactive Base Excision Repair (BER) pathway, with existing or newly developed inhibitors, could be beneficial to resensitize resistant GBM to the DNA alkylation damage of TMZ. Methods and Materials Patient-derived Brain Tumour Initiating Cells (BTICs) were used as our GBM model. BTICs were cultured and treated with increasing concentrations of Temozolomide (TMZ) alone or in combination with various inhibitors targeting the Base Excision Repair (BER) pathway. DNA damage was assessed using our novel high-throughput alkaline 3-D comet assay, while self-renewal and proliferation capacities were evaluated through sphere formation, cell viability, and serial-replating assays. Results A novel high-throughput alkaline 3D comet assay was developed and optimized to measure DNA damage specifically in BTICs. Using this assay, resistant BTICs were re-sensitized to TMZ damage with BER inhibitors Pamiparib, Olaparib, and the novel compound B9 which targets the scaffolding protein XRCC1 within the BER. Self-renewal and cell proliferation assays confirmed that BER inhibition impaired growth and sphere-formation capacity of resistant BTICs when combined with TMZ. Serial re-plating assay showed that BER inhibitors reduced long-term self-renewal and proliferation potentials of TMZ treated BTICs, as they failed to form spheres and showed reduced proliferation after being moved to drug-free conditions post-treatment. Conclusion This study shows that targeting the BER repair pathway can effectively re-sensitize TMZ-resistant BTICs, which provides a promising strategy to overcome therapeutic resistance in GBM. Our Novel compound B9 shows significant potential, making XRCC1 inhibition and biomarker analysis important objectives of our work. Furthermore, our novel high-throughput 3D comet assay platform represents a valuable tool for DNA damage analysis in tumorspheres, which could facilitate large-scale drug screening in GBM as well as other diseases.