Matrix-assisted laser desorption ionization-mass spectrometry is an efficient analytical method for large-scale identification of proteins separated by two-dimensional polyacrylamide gel electrophoresis. Following in-gel digestion, the salt present in the peptide extracts is usually removed by chromatography prior to analysis. Desalting is a labor-intensive and time-consuming step, limiting the total number of samples that can be processed daily. We improved the daily sample output by performing the in-gel protein digestion in low-salt, nonvolatile buffer and simplifying the recovery of the generated peptides, collecting them in a small volume by sonication. This technique is routinely used for identification of proteins of Haemophilus influenzae and human brain. The methodology described facilitates the analytical process and allows the analysis of hundreds of proteins per day. Furthermore, it represents an essential step toward process automation.