Mass spectrometry is a powerful technique for the identification of proteins at nanogram quantities. However, some degree of sample preparation prior to mass spectrometry is required, and silver‐stained protein gel samples are most problematic. Here we report our strategy to obtain peptide mass profiles from silver‐stained protein gel samples from one‐ or two‐dimensional gels by destaining prior to enzymatic digestion. This study demonstrates that by using the destaining method, the sensitivity and quality of mass spectra is increased for matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometric analysis, permitting more proteins to be identified by peptide mass database analysis.