Acetylation of proteins on specific lysine residues by acetyltransferase enzymes is a post-translational modification for biologically relevant regulation. In this study, we proposed a strategy to determine the in vitro acetylation sites of proteins by tracing isotope-labeled acetyl groups using mass spectrometry. Isotope-labeled and unlabeled acetyl groups transferred onto the substrates in vitro result in a specific "mass difference" that can be measured by MS analysis and utilized for localization of potential acetylated peptide signals. The identification of acetylation site is facilitated by conducting MS/MS experiments on those selected signals. Acetylation reactions of substrates were performed in the presence of acetyltransferase and equal molar of isotope-labeled acetyl coenzyme A ([(13)C2-2-D3]-acetyl-CoA) and unlabeled acetyl-CoA. After enzymatic digestion, the resulting peptide mixture was fractionated by off-line, reversed-phase high-pressure liquid chromatography and the accurate mass measurement of peptides was achieved by a quadrupole/time-of-flight mass spectrometer. Signals with 5-Da (or their multiples) mass differences and equal responses were selected out by program computation. Those potential acetylated peptide signals were subjected to MS/MS analyses for determination of acetylation sites. We have used histone H3 peptide (aa 1-20), histone H2B peptide (aa 1-21), histone H2A, and histone H2B proteins as the model compounds to demonstrate the applicability of this analytical scheme for the characterization of in vitro acetylation sites.