Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl groups from the epsilon-amino groups of conserved lysine residues in the amino terminal tail of histones. In humans, there are 18 potential deacetylase enzymes that are responsible for the removal of acetyl groups and maintenance of the equilibrium of lysine acetylation on histones. Like most histone modification enzymes, accumulating evidence suggests that many, if not all, HDACs can also modify non-histone proteins. The focus of this article is to provide up-to-date, easy to follow, approaches and techniques specifically for the assay of HDAC enzymatic activities.