Abstract INTRODUCTION Fluorescence-guided-surgery can potentially facilitate maximal safe resection of high-grade-gliomas. 5-aminolevulinic-acid (5-ALA) leads to enzymatic accumulation of protoporphyrin-IX (PpIX) in high-grade neoplastic cells. A high-dose infusion of indocyanine-green (ICG) (Second-Window-ICG or SWIG) relies primarily on the passive accumulation of ICG in neoplastic tissue via enhanced-permeability-and-retention. In this study, we compare 5-ALA and SWIG in a murine allograft HGG model and human patients with HGG. METHODS Six-week-old C57/BL6 mice underwent intracranial implantation of GL261 cells and received 5 mg/kg ICG (24-h pre-euthanasia) and 200 mg/kg 5-ALA (3-h pre-euthanasia) once tumors developed. Fresh brain coronal sections were imaged for PpIX and ICG fluorescence and H&E stains were performed on fixed specimens. The fluorescence images and H&E images were overlaid and multiple region-of-interest (ROIs) were sampled at random to calculate the receiver operating-characteristic for each fluorophore. Furthermore, 3 patients with HGG were administered 5 mg/kg ICG 24-h preoperatively and 20 mg/kg 5-ALA 3-h preoperatively. Intraoperatively, tumor specimens were imaged for both ICG and PpIX fluorescence. RESULTS Four-hundred ROIs were sampled from 25 coronal slices (217 neoplastic). PpIX fluorescence signal-to-background (SBR) was significantly higher in neoplastic ROIs (2.21 vs 1.33, P-value < .0001). 5-ALA accuracy was 79.1% (95% confidence interval 74.5-83.6%). SWIG SBR was significantly higher for neoplastic ROIs (4.25 vs 1.91, P-value < .0001). SWIG accuracy was higher than 5-ALA’s at 88.2% (95% confidence interval 84.7-91.7%) (P-value < .0001). The optimal SBR cutoffs for 5-ALA and SWIG were 1.5 and 2.15, respectively. The most distinct difference between 5-ALA and SWIG distribution was seen in areas of necrosis/hemorrhage, which often showed weak/no PpIX fluorescence but strong SWIG fluorescence. 5-ALA accuracy was lower in necrotic tumors than in non-necrotic tumors (67.8% vs 86.3%, P-value = .0001). In 3 HGG patients who received both 5-ALA and SWIG, 17 biopsy specimens were obtained (15 neoplastic). Near-infrared imaging revealed 14 true-positive, 2 true-negative, and 1 false-negative signals, while PpIX imaging revealed 12 true-positives, 1 true-negative, 1 false-positive, and 3 false-negative signals. CONCLUSION In this first comparative study of 5-ALA and SWIG, we demonstrate similar diagnostic utility of a passive permeability dye (ICG) to an enzymatic functional dye (5-ALA) in HGG. Considering the benefits of near-infrared fluorophores over visible-spectrum fluorophores, further comparative studies in clinical settings are warranted.