This chapter highlights acylaminoacyl-peptidase enzyme, which catalyzes the removal of a blocked amino acid from a blocked peptide. The products of the reaction are an acyl amino acid and a peptide with a free N terminus shortened by one amino acid. The enzyme acts on a variety of substrates, including peptides with different N-terminal acyl groups, such as acetyl, chloroacetyl, formyl, and carbamyl groups. The optimum length of the blocked peptide substrate is 2-3 amino acids, but larger peptide substrates are also cleaved at slower rates. Acylaminoacyl-peptidase can also act on nascent peptides during biosynthesis and hydrolyze bioactive peptides. For purification of the enzyme, activity is most easily determined with acetylalanine p-nitroanilide as substrate. Asit is fast, sensitive, and easy to perform, this assay is used for monitoring fractions collected during column chromatography. The rate of formation of product, p-nitroaniline, is followed at 405 nm as a function of time. The enzyme is inhibited by several types of reagents, including diisopropyl fluorophosphate (DFP), p-hydroxymercuribenzoate, and some heavy metals, such as Hg2+and Cd2+.