A simple, fast, sensitive and inexpensive UV-spectrophotometric method for the determination of amoxicillin in pharmaceutical preparations has been developed based on two enzymatic reactions. In this method, D-4-hydroxyphenylglycine side chain of amoxicillin was selectively cleaved off by penicillin acylase. Subsequently. it was reacted with 2-oxoglutarate. by the catalysis Of D-phenylglycine aminotransferase, to yield the product with high UV absorption namely 4-hydroxybenzoylformate. The amount of amoxicillin was then determined as a change in absorbance at 335 nm. In this work, the assay conditions were studied and optimized and the method was validated. The calibration curve presented an excellent linearity with r(2) of 0.9998 (0-100 mu M amoxicillin). Detection and quantitation limits were 0.77 and 2.55 mu M, respectively. Good accuracy and precision were obtained when the method was tested with amoxicillin capsules and powder for oral suspension. No interference from common excipients in the formulations or degradation products was observed. Finally, since all procedures were performed without the use of any organic solvents or hazardous chemicals which were detrimental to the environment and had a low consumption of reagents. this proposed assay was an ideal green analytical method suitable for the quality control of amoxicillin in pharmaceuticals. (C) 2009 Elsevier Inc. All rights reserved.