Abstract 6-( N-Acetyl-L-phenylalanyl)-aminoluciferin as an example for a new class of highly sensitive bioluminogenic substrates that has been synthesized and characterized. These peptide derivatives can be used regarding to the peptide residue as highly sensitive substrates for different proteinases. Here we demonstrate the use of 6-( N-acetyl-L-phenylalanyl)-aminoluciferin as a novel bioluminogenic substrate for α-chymotrypsin. The kinetic parameters determined for 6-( N-acetyl-L-phenylalanyl)-aminoluciferin are K m = 0.38 mmol/liter, k cat = 6.5 s −1, and k cat/ K m = 17,100 (liter/mol·s). The test principle of the coupled bioluminogenic assay is the release of aminoluciferin by enzymatic cleavage of 6-( N-acetyl-L-phenylalanyl)-aminoluciferin. Aminoluciferin is a very sensitive substrate of firefly luciferase and can be easily quantified in a luminometric assay. Amounts of chymotrypsin down to 0.3 ng per assay can be routinely determined.