To complement a project towards the development of real-time optical biopsy for brain tissue discrimination and surgical resection guidance, the optical properties of various brain tissues were measured in vitro and correlated to features within clinical diffuse reflectance tissue spectra measured in vivo. Reflectance and transmission spectra of in vitro brain tissue samples were measured with a single-integrating-sphere spectrometer for wavelengths 400–1300 nm and converted to absorption and reduced scattering spectra using an inverse adding-doubling technique. Optical property spectra were classified as deriving from white matter, grey matter or glioma tissue according to histopathologic diagnosis, and mean absorption and reduced scattering spectra were calculated for the three tissue categories. Absolute reduced scattering and absorption values and their relative differences between histopathological groups agreed with previously reported results with the exception that absorption coefficients were often overestimated, most likely due to biologic variability or unaccounted light loss during reflectance/transmission measurement. Absorption spectra for the three tissue classes were dominated by haemoglobin absorption below 600 nm and water absorption above 900 nm and generally determined the shape of corresponding clinical diffuse reflectance spectra. Reduced scattering spectral shapes followed the power curve predicted by the Rayleigh limit of Mie scattering theory. While tissue absorption governed the shape of clinical diffuse reflectance spectra, reduced scattering determined their relative emission intensities between the three tissue categories.