A rapid and accurate spectrophotometric assay has been developed for the determination of blood werum levels of gentamicin and related antibiotics. The assay uses a purified enzyme from Escherichia coli JR88/C600 that acetylates gentamicin with the production of coenzyme A, linked to a chemical reaction with a sulfhydryl reagent to produce stoichiometric amounts of a sensitive chromophore, monitored in the visible spectrum. The system provides advantages of speed, cost, convenience, accuracy, and enzyme stability to the desirable characteristics encountered with previous enzymatic methods.