Most preclinical studies on glioblastoma (GBM) fail to provide translational utility in the clinic. Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) improves tumor resection, disease prognosis, and thus, patient outcomes. Given the critical role of surgery in managing recurrent GBM, it is essential to incorporate surgical elements into preclinical models to accurately reflect clinical scenarios and enhance translational success. However, existing protocols for 5-ALA-guided resection in preclinical models are limited and often lack clinical relevance. To address this gap, we developed a novel protocol for the 5-ALA-guided resection in two mouse GBM models: TRP-mCherry-FLuc and GL261 Red-FLuc. Resection of TRP-mCherry-FLuc tumors significantly extended survival and mitigated weight loss compared to controls. Similarly, GL261 Red-FLuc tumor resection increased survival, reduced weight loss, and slowed tumor progression. This study presents a clinically relevant protocol for 5-ALA-guided resection in preclinical GBM models, providing a platform for future research to integrate adjuvant therapies and enhance their potential translation into clinical practice.