Huntingtin (Htt) is a protein with a polyglutamine stretch in the N-terminus and expansion of the polyglutamine stretch causes Huntington's disease (HD). Htt is a multiple domain protein whose function has not been well characterized. Previous reports have shown, however, that post-translational modifications of Htt such as phosphorylation and acetylation modulate mutant Htt toxicity, localization, and vesicular trafficking. Lysine acetylation of Htt is of particular importance in HD as this modification regulates disease progression and toxicity. Treatment of mouse models with histone deacetylase inhibitors ameliorates HD-like symptoms and alterations in acetylation of Htt promotes clearance of the protein. Given the importance of acetylation in HD and other diseases, we focused on the systematic identification of lysine acetylation sites in Htt23Q (1-612) in a cell culture model using mass spectrometry. Myc-tagged Htt23Q (1-612) overexpressed in the HEK 293T cell line was immunoprecipitated, separated by SDS-PAGE, digested and subjected to high performance liquid chromatography tandem MS analysis. Five lysine acetylation sites were identified, including three novel sites Lys-178, Lys-236, Lys-345 and two previously described sites Lys-9 and Lys-444. Antibodies specific to three of the Htt acetylation sites were produced and confirmed the acetylation sites in Htt. A multiple reaction monitoring MS assay was developed to compare quantitatively the Lys-178 acetylation level between wild-type Htt23Q and mutant Htt148Q (1-612). This report represents the first comprehensive mapping of lysine acetylation sites in N-terminal region of Htt.