Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver‐ and Coomassie‐staining protocols with a nano‐LC‐MS/MS system was systematically elucidated. Altogether, 13 different silver‐staining, 1 imidazole‐staining and 2 Coomassie‐staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L‐lactate dehydrogenase, bovine milk β‐lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver‐staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93–99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850–858) combine good sequence coverage and good sensitivity and are recommended for nano‐LC‐MS/MS analysis.