Extent of resection is a major goal and prognostic factor in the treatment of gliomas. In this study we evaluate whether quantitative ex vivo tissue measurements of δ-aminolevulinic acid-induced protoporphyrin IX (PpIX) identify regions of increasing malignancy in low- and high-grade gliomas beyond the capabilities of current fluorescence imaging in patients undergoing fluorescence-guided resection (FGR). Surgical specimens were collected from 133 biopsies in 23 patients and processed for ex vivo neuropathological analysis: PpIX fluorimetry to measure PpIX concentrations (C(PpIX)) and Ki-67 immunohistochemistry to assess tissue proliferation. Samples displaying visible levels of fluorescence showed significantly higher levels of C(PpIX) and tissue proliferation. C(PpIX) was strongly correlated with histopathological score (nonparametric) and tissue proliferation (parametric), such that increasing levels of C(PpIX) were identified with regions of increasing malignancy. Furthermore, a large percentage of tumor-positive biopsy sites (∼40%) that were not visibly fluorescent under the operating microscope had levels of C(PpIX) greater than 0.1 µg/mL, which indicates that significant PpIX accumulation exists below the detection threshold of current fluorescence imaging. Although PpIX fluorescence is recognized as a visual biomarker for neurosurgical resection guidance, these data show that it is quantitatively related at the microscopic level to increasing malignancy in both low- and high-grade gliomas. This work suggests a need for improved PpIX fluorescence detection technologies to achieve better sensitivity and quantification of PpIX in tissue during surgery.