The analytical performance of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for direct analysis of peptide and protein mixtures is strongly dependent on the sample and matrix preparation. A two-layer sample preparation method is demonstrated to be very effective for analyzing complex mixtures. In this method, the first layer on the MALDI probe is the densely packed matrix microcrystals formed by fast solvent evaporation of a matrix solution. A mixture solution containing both matrix and sample is then deposited onto the first layer to form uniform analyte/matrix micrococrystals. It is found that the addition of matrix to the second-layer sample solution proves to be critical in analyzing mixtures of peptides and proteins covering a broad mass range. The effect of solvent conditions for preparing the second-layer solution is discussed. The application of this method is demonstrated for the analysis of cow's milk where milk proteins as well as peptide fragments produced from proteins by indigenous proteinases are detected. Direct analyses of peptides and proteins from a bacteria extract and crude egg white are also illustrated.