Retinal nerve fiber layer is the innermost layer of the retina, it is formed of the axons of ganglion cells. Attrition of nerve fibers in the anterior visual pathways is reflected directly in the nerve fiber layer of the retina. These retrograde degenerative changes are subtle but they can be seen with an ophthalmoscope and can be photographed with a fundus camera. So, the importance of the new objective quantitative technologies for RNFL assessment which are the optical coherence tomography, the confocal scanning laser ophthalmoscopy, the scanning laser polarimetry and the retinal thickness analyzer had been proved. Glaucoma is a multi-factorial optic neuropathy characterized by irreversible progressive loss of the retinal ganglion cells (RGCs) and thinning of the retinal nerve fiber layer (RNFL), leading to visual field loss and eventually, total loss of vision. 40% to 50% of the RNFL could be lost before visual field defects are detected by conventional perimetry. Thus, RNFL assessment had emerged as an important parameter for pre-perimetric diagnosis of glaucoma. Diabetic retinopathy (DR) is the fifth most common cause of blindness worldwide. In addition to typical retinopathies, neuro-visual impairments had been reported. Focal retinal nerve fiber layer loss in diabetic patients with preclinical DR was detected. In vitro studies had demonstrated that diabetes affects both retinal neurons and glial cells. Panretinal photocoagulation causes a sequential decrease in the peripapillary retinal nerve fiber layer (RNFL) thickness. On the other hand, some diseases had been thought to change RNFL, but studies had proved the opposite and that aided in the true understanding of these pathologies as in amblyopia in which RNFL assessment was insignificant.