Current proteomic technologies can effectively be used to study the proteins of the vitreous body and retina in health and disease. The use of appropriate samples, analytical platform and bioinformatic method are essential factors to consider when undertaking such studies. Certain proteins may hinder the detection and evaluation of more relevant proteins associated with pathological processes if not carefully considered, particularly in the sample preparation and data analysis stages. The utilization of more than one quantification technique and database search program to expand the level of proteome coverage and analysis will help to generate more robust and worthwhile results. This review discusses important aspects of sample processing and the use of label and label-free quantitative proteomics strategies applied to the vitreous and retina.