Abstract INTRODUCTION Fluorescence with 5-aminolevulinic acid (5-ALA) can be used as an intraoperative marker of malignant glial tumor tissue in brain biopsies.However, there is a scarcity of data regarding its real benefit. This study aims to evaluate the safety and accuracy of this diagnostic tool. METHODS Single-center prospective analysis of patients with suspected high-grade brain tumors diagnosed by 5-ALA guided frameless stereotactic biopsy. RESULTS A total of 198 biopsies qualified for final analysis. Biopsies of high-grade primary brain tumors showed positive rates of fluorescence in 94.28% of cases (P < .01). No clinical adverse effects associated with use of 5-ALA were observed. CONCLUSION Study findings suggest that the administration of 5-ALA fluorescence can use safety and have a high accuracy as an intraoperative diagnostic tool in high-grade glioma.