Histone acetyltransferases (HATs) are important chromatin modifying enzymes that catalyze acetylation of specific lysine residues in histone and nonhistone substrates. They participate in multiple cellular processes such as transcriptional regulation and signal transduction. Aberrant expression of HATs has been observed in various disease states, especially cancer. However, current strategies used for studying HAT enzymatic activity and inhibitor discovery are quite limited. We report here a novel strategy for the homogeneous, continuous, one-step measurement of HAT activity. A series of fluorescent reporters based on the amino-terminal seqence of histone H4 were synthesized and evaluated for HAT assay. Fluorescence signals change effectively in response to the acetylation process by HAT p300. Such an assay should thus be broadly useful for assaying HAT activity in vitro as well as valuable in discovering new anticancer drugs based on the modulation of the HAT targets.