The combinations of gel electrophoresis or liquid chromatography (LC) and mass spectrometry are the two popular approaches for large-scale protein identification. However, these two approaches are both limited by their inability to speed up the protein digestion process. Present researches for fast protein enzymatic digestion mainly focused on known proteins. Whether they can be used to protein mixtures is not well addressed. In this paper microwave technology is used to develop a fast protein preparation and enzymatic digestion method for protein mixtures. The protein mixtures in solution or in gel were prepared and digested by microwave-assisted protein enzymatic digestion (MAPED) method, which could rapidly produce peptide fragments. The peptide fragments were further analyzed by capillary LC and electrospray ion trap mass spectrometry (ESI-IT-MS) or matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The efficacy of this technique was developed by bovine serum albumin and retested by human urinary proteins and yeast lysate. The method enabled to prepare and digest protein mixtures in solution (human urinary proteins) or in gel (yeast lysate) in 6 min or 25 min, and could obtain the same (in-solution) or better (in-gel) peptide yield comparing with standard overnight digestion method. This new application of microwave technology to protein mixtures preparation and enzymatic digestion will fasten the application of proteomics techniques to biological and clinical researches.