Histone acetyltransferases (HATs) have critical roles in cellular functions and are involved in the regulation of gene transcription, differentiation, and proliferation. The HAT Activity Colorimetric Assay Kit offers a convenient, non-radioactive system for a rapid and sensitive detection of HAT activity in mammalian samples. The kit includes an active Nuclear Extract (NE) to be used as a positive control, HAT cofactor, acetyl-CoA, and all downstream cofactors and substrates. Acetylation of a peptide substrate by active HAT releases the free form of CoA, which then serves as an essential coenzyme for producing NADH. NADH can easily be detected spectrophotometrically upon reacting with a soluble tetrazolium dye. The detection can be continuous, and therefore, is suitable for kinetic studies. The kit provides all reagents and a simple, straightforward protocol for a complete assay.