5-Aminolevulinic acid (5-ALA)-mediated fluorescence does not effectively depict low grade gliomas (LGG) or the infiltrative tumor portion of high-grade gliomas (HGG). While spectroscopy improves sensitivity and precision, this is currently limited by autofluorescence and a second protoporphyrin IX (PpIX) fluorescence state at 620 nm. We investigated the autofluorescence to better characterize the present spectra and thus increase PpIX quantification precision and sensitivity. This study included 128 patients undergoing surgery for malignant glioma. 5-ALA (Gliolan) was administered before anesthesia, and fluorescence was measured using a hyperspectral device. It was found that all 2692 measured spectra consisted of contributions from 620 to 634 nm PpIX, NADH, lipofuscin, and flavins. The basis spectra were characterized and their use in spectral unmixing led to 82.4% lower fitting error for weakly fluorescing areas (p