The invention provides methods and kits for characterizing the activity of an acetyl transferase or deacetylase. The method involves enzymatically acetylating or deacetylating in vitro a substrate that is a peptide fragment of a full-length polypeptide, and then non-enzymatically acylating the peptide substrate with acyl groups that differ in molecular weight from the enzymatically added or removed acetyl groups. Typically, deuterated acetic anhydride is used to non-enzymatically acylate the substrate. The fully acylated substrate is then characterized by mass spectrometry to determine the amino acid positions of the substrate that are enzymatically acetylated or deacetylated.