Organic matter contained in food waste was degraded by anaerobic digestion under mesophilic and thermophilic conditions at two hydraulic retention times. Evolution of the digestion process was followed by thermogravimetry analysis, fluorescence spectroscopy and (1)H nuclear magnetic resonance. All analytical methods suggested that longer retention times might be required for food waste stabilization under mesophilic conditions as compared to thermophilic stabilization. All the analytical methods showed that the stabilization process consisted of two steps, where complex organic molecules were formed during initial stabilization and then digested providing sufficient hydraulic retention time. Longer hydraulic retention times were required for food waste stabilization under mesophilic conditions. Overall, thermal and (1)H NMR analyses of the digestate samples might be recommended if more detailed analysis is required, while fluorescence measurements can be used as a fast screening technique, which provides qualitative assessment of the stabilization process.