Lysine acetylation is an important posttranslational modification known to alter protein structure and function. Understanding the mechanisms involved in regulating protein acetylation remains a key factor in elucidating what role this modification plays in numerous disease pathologies. Here, we describe an in vitro strategy to examine the site-specific deacetylation of proteins utilizing the chemical acetylation of protein lysine residues via acetic anhydride. The impact of chemical acetylation on protein lysine residues is characterized by native gel electrophoresis and Western blotting. Acetyl-Lys modifications are then examined for deacetylation using a SIRT3 deacetylase activity assay and followed by stable isotope dilution mass spectrometry.