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Histone deacetylase (HDAC) inhibitors are currently used in anticancer therapy to perturb genomic targets involved in gene transcriptional responses. However, the role of HDAC inhibitors on the acetylation of proteins outside of the transcriptional network has not been thoroughly assessed. We recently discovered that one of the HDACs, HDAC3, is localized on the mitotic spindle and regulates proper mitotic progression (1). To determine potential HDAC targets, we undertook a proteomics approach to search for acetylated proteins in mitosis (2). First, we synchronized cells in mitosis and used a polyclonal anti-acetyl-Lysine antiserum to immunoprecipitate acetylated proteins, followed by their identification by LC-ESI-MS/MS. We then confirmed the acetylation status of several mitotic proteins by anti-acetyl-Lysine immunoprecipitation with a monoclonal antibody followed by Western blot analyses of the proteins of interest. We further confirmed by a reciprocal immunoprecipitation with protein-specific antibody followed by Western blot analysis with another monoclonal anti-acetyl-Lysine antibody. Interestingly, the acetylation of a subset of the mitotic proteins can be further enhanced by treatment with apicidin, a small molecule inhibitor with specificity for HDAC3, suggesting that their acetylation may be regulated by HDAC3 in mitosis. In this chapter, we describe the various techniques using NudC as an example of an acetylated protein that is sensitive to apicidin treatment in mitosis.