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Simple Summary: This study explores the use of fluorescence-guided resection in glioblastoma surgery, focusing on the combined use of 5-aminolevulinic acid and sodium fluorescein. By analyzing 100 cases from our medical center, we aimed to address concerns about fluorescence-guided resection and share our findings. The dual use of 5-aminolevulinic acid and fluorescein enhances the extent of tumor resection and reduces false positives without increasing adverse effects. In our experience, fluorescein guided the initial resection phase, while 5-aminolevulinic acid identified tumor spots within the surgical cavity, achieving gross total resection in 96% of cases and supra-maximal resection in 11%. This combined approach appears promising for improving outcomes in glioblastoma patients. Background: Fluorescence-guided resection (FGR) of glioblastomas has been previously explored with the use of 5-amivelulinic acid (5-ALA) and sodium fluoresceine (SF), allowing us to maximize the extent of resection (EoR). In this study, we highlight the most relevant concerns regarding this technique and present the methods and results from the experience of our center. Methods: A case series of 100 patients operated on in AOU Città della Salute e della Scienza in Turin with a histological diagnosis of glioblastoma (grade IV, according to WHO 2021) was retrospectively analyzed. Both 5-ALA and SF were administered and intraoperatively assessed with an optical microscope. Results: 5-ALA is the only approved drug for FGR in glioblastoma, reporting an increased EoR. Nevertheless, SF can be positively used in addition to 5-ALA to reduce the risk of false positives without increasing the rate of adverse effects. In our experience, SF was used to guide the initial phase of resection while 5-ALA was used to visualize tumor spots within the surgical cavity. In 96% of cases, gross total resection was achieved, with supra-maximal resection in 11% of cases. Conclusions: Combined FGR using 5-ALA and SF seems to be a promising method of increasing the extent of resection and to improving the prognosis in glioblastoma patients. [ABSTRACT FROM AUTHOR]