A combination of nanoelectrospray tandem mass spectrometry and O-18-labeled peptide internal standards was applied for the absolute quantification of proteins from their in-solution and in-gel tryptic digests. Although absolute quantification from in-solution digests was accurate, we observed that in-gel digestion compromised the quantification accuracy by affecting the recovery of individual peptides and, therefore, the provided estimates might be strongly influenced by the selection of reference peptides. Under optimized experimental conditions, it was possible to provide a semiquantitative estimate of the absolute amount of gel separated proteins within better than 50% error margin.