To provide a substrate for measuring histone deacetylase (HDAC) activity which enables a nonradioactive measurement of HDAC, identification of HDAC subtypes and also screening of inhibitors specific for respective subtypes, which has been considered to be impossible, and also to provide a method for measuring the enzymatic activity of HDAC and a method for screening the HDAC inhibitors using the substrate.

A compound which contains a neighboring sequence of an acetylated part of histone H4, p53, or the like, and is represented by formula (I) (wherein, R1 is H or a protective group of an amino terminal; R2 is a group which is deacetylated by HDAC; R3 is a fluorescent or a chromophoric group which forms an amide bond between the carbonyl terminal of a lysine and changes its fluorescent or chromophoric characteristics in response to free amine generated from the hydrolysis of the amide bond; X is one or more amino acid residues; and (K) is a lysine residue) is used as the substrate for measuring the HDAC activity.