Vertebrate retinas have several characteristics that make them particularly interesting from a metabolic perspective. The retinas have a highly laminated structure, high energy demands, and they share several metabolic features with tumors, such as a strong Warburg effect and abundant pyruvate kinase M2 isoform expression. The energy demands of retinas are both qualitatively and quantitatively different in light and darkness and metabolic dysfunction could cause retinal degeneration. Stable isotope-based metabolic analysis with mass spectrometry is a powerful tool to trace the dynamic metabolic reactions and reveal novel metabolic pathways within cells and between cells in retina. Here, we describe methods to quantify retinal metabolism in intact retinas and discuss applications of these methods to the understanding of neuron-glia interaction, light and dark adaptation, and retinal degenerative diseases.