A comparison of automated in-gel digestion methods for low picomolar to femtomolar levels of protein is presented. Gel spots with 4 pmol to 120 fmol of protein were stained with either Coomassie colloidal blue or SYPRO Ruby and digested using an automated platform. The sequence coverages and average peak intensities obtained from a matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis are compared. Results show that methods using an acetonitrile extraction or digest times greater than the standard 4 h give no significant increase in peptide sequence coverage for automated digestion of low protein level samples. It is also shown that digests from SYPRO Ruby-stained gels show a greater improvement upon ZipTip cleanup than digests from Coomassie colloidal blue-stained gels. The digests from SYPRO Ruby-stained gels are also shown to give a higher average peptide intensity if a method with minimal gel plug washing is used.