A novel method for assaying the substrate specificity of proteolytic enzymes has been developed utilizing ligand-enhanced lanthanide ion fluorescence. This approach was used to develop peptide libraries to probe substrate specificity in the prime sites of proteolytic enzymes. A positional scanning synthetic combinatorial library of fluorogenic peptides was synthesized and used to determine the extended prime site specificity of bovine alpha-chymotrypsin. The enzyme showed a preference for Lys and Arg in the P1' position, rather broad specificity in the P2' position, and a slight Arg specificity in the P3' position. The specificity profile of bovine alpha-chymotrypsin agrees well with previously reported data, and the substrate library reported herein should provide valuable information about the prime site substrate specificities of other proteolytic enzymes as well. Furthermore, the continuous fluorogenic assay described may prove useful in analyzing the activity of other hydrolytic enzymes.