Publisher Summary This chapter describes a variety of methods that have been useful in analyzing the activity and regulation of hGcn5, including methods to prepare recombinant hGcn5 or endogenous hGcn5 fractionated from HeLa nuclear extract. Protocols for assaying the phosphorylation of hGcn5 by DNA-dependent protein kinase (DNA-PK) and for determining the effect on the histone acetyltransferase (HAT) activity of hGcn5, both in vivo and in vitro, are discussed in the chapter. These approaches are useful in analogous studies of the steadily increasing number of HATs and histone deacetylases (HDACs), as well as in analyzing other posttranslational modifications that may alter activity. Correct refolding and enzymatic activity of hGcn5 is verified by two methods. First, hGcn5 is shown to have correct structure by demonstrating specific interaction with hAda2 protein—for example, GST–hGcn5 interacts physically with in vitro translated [35S]hAda2. Second, His6-hGcn5 possesses enzymatic HAT activity on free-histone substrates. The chapter also discusses the effect of phosphatase treatment on recombinant hGcn5.