As proteomic technology evolves, protein staining sensitivity is constantly being improved, enabling researchers to better visualize the proteome of their system. The current challenge is to balance the limits of detection of protein visualization with those of the mass spectrometric methods. In this report, mass spectra generated from human serum or rat liver proteins stained with either colloidal Coomassie blue, Daiichi silver, SYPRO Orange, SYPRO Red, SYPRO Ruby, or SYPRO Tangerine are compared. It has been concluded that the newest generation of fluorescent protein stains, compared with traditional staining methods, are more compatible to matrix‐assisted laser desorption/ionization (MALDI) and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) methods. The number of database matches obtained using each mass spectrometry method and the percent sequence coverage obtained from trypsin digested proteins stained using these six methods is provided.