Abstract: A rapid method for the specific detection of the presence of Beta-lactamase from microbial sources is disclosed. The method utilizes a Beta-lactam ring containing substrate whose amide bond is hydrolyzed in the presence of Beta-Lactamase. A substrate, which includes a Beta-Lactam antibiotic with an acyl side chain containing an .alpha.-amino group and a .alpha.-phenyl group and/or its derivatives is first contacted with an organism thought to produce Beta-lactamase or a cell-free Beta-lactamase preparation, and, subsequently, it is determined whether the reaction product between the substrate and the organism or the preparation fluoresces.