The objective of this in vitro tissue study is to investigate the feasibility of using optical spectroscopy to differentiate pediatric neoplastic and epileptogenic brain from normal brain. Specimens are collected from 17 patients with brain tumors, and from 26 patients with intractable epilepsy during surgical resection of epileptogenic cerebral cortex. Fluorescence spectra are measured at excitations of 337, 360, and 440 nm; diffuse reflectance spectra are measured between 400 and 900 nm from each specimen. Pathological analysis is performed to classify abnormalities in brain specimens, and its findings are correlated with spectral data. Statistically significant differences (p<0.01) are found for both raw and normalized diffuse reflectance and fluorescence spectra between 1. neoplastic brain and normal gray matter, 2. epileptogenic brain and normal gray matter, and 3. neoplastic brain and normal white matter. However, no distinct spectral features are identified that effectively separate epileptogenic brain from normal white matter. The outcomes of the study suggest that certain unique compositional and structural characteristics of pediatric neoplastic and epileptogenic brain can be detected using optical spectroscopy in vitro.