Quantitative fundus autofluorescence (qAF) is an approach that is built on a confocal scanning laser platform and used to measure the intensity of the inherent autofluorescence of retina elicited by short-wavelength (488 nm) excitation. Being non-invasive, qAF does not interrupt tissue architecture, thus allowing for structural correlations. The spectral features, cellular origin and topographic distribution of the natural autofluorescence of the fundus indicate that it is emitted from retinaldehyde-adducts that form in photoreceptor cells and accumulate, under most conditions, in retinal pigment epithelial cells. The distributions and intensities of fundus autofluorescence deviate from normal in many retinal disorders and it is widely recognized that these changing patterns can aid in the diagnosis and monitoring of retinal disease. The standardized protocol employed by qAF involves the normalization of fundus grey levels to a fluorescent reference installed in the imaging instrument. Together with corrections for magnification and anterior media absorption, this approach facilitates comparisons with serial images and images acquired within groups of patients. Here we provide a comprehensive summary of the principles and practice of qAF and we highlight recent efforts to elucidate retinal disease processes by combining qAF with multi-modal imaging.