Metabolomics refers to the systematic identification and quantification of the small molecule metabolites (the metabolome) of a biological system in a given space (cell, tissue, organ, biological fluid, or organism) and time. Global metabolic profiling provides broad range of coverage for most of the analytes present in any tissue. Human retina is metabolically highly active, and retinal vascular diseases such as age-related macular degeneration (AMD), diabetic retinopathy (DR), retinal vein occlusion (RVO), central retinal artery occlusion, and retinopathy of prematurity (ROP) are often associated with the disruptions in metabolic activities. A systematic study of total retinal metabolites from human diseased retina is a major challenge owing to the nonavailability of tissue specimens. Therefore, vitreous humor being very proximal to retina could be used as surrogate for retinal metabolomic analysis. As the extraction method adopted for such analysis determines the type of metabolites, two different types of solvent (methanol and chloroform)-based extraction methods could be used for retinal vascular patient samples (vitreous humor). Metabolites obtained from both the extraction methods are then subjected to LC-MS/MS for detection and identification.