The synthesis of 2-nitro-5-[(hexanoyl)-amino]-benzoic acid and 2-nitro-5-[(octanoyl)-amino]-benzoic acid as chromogenic substrates for the determination of aliphatic penicillin acylase activity is described. During enzymatic hydrolysis, the released chromophore, 2-nitro-5-amino-benzoic acid, was detected at 405 nm. Penicillin acylase from Streptomyces lavendulae had an appreciable activity towards these substrates, which can then be used to detect penicillin acylases able to cleave hexanoyl and octanoyl residues off synthetic amides as well as penicillin dihydroF and penicillin K, their natural analogues.