Fluorescence and diffuse reflectance spectroscopy are powerful tools to differentiate normal and malignant tissue based on the emissions from endogenous fluorophores and diffuse reflection of absorbers such as hemoglobin. However, separate analytical methods are used for the identification of fluorophores and hemoglobin. The estimation of fluorophores and hemoglobin simultaneously using a single technique of autofluorescence spectroscopy is reported, and its diagnostic potential on clinical tissue samples is potentially exploited. Surgically removed brain tissues from patients that are later identified pathologically as astrocytoma, glioma, meningioma, and schwannoma are studied. The emissions from prominent fluorophores collagen, flavin adenine dinucleotide, phospholipids, and porphyrin are analyzed at 320 and 410 nm excitations. The hemoglobin concentration is also calculated from the ratio of fluorescence emissions at 500 and 570 nm. A better classification of normal and tumor tissues is yielded for 410 nm excitation compared to 320 nm when diagnostic algorithm based on linear discriminant analysis is used. The potential of fluorescence spectroscopy as a single entity to evaluate the prominent fluorophores as well as the hemoglobin concentration within normal and tumor brain tissues is emphasized.