The reaction ofd,l-7-azatryptophan (d,l-7AW) with tryptophanyl-tRNA synthetase (TrpRS), adenosine triphosphate (ATP), and Mg2+in the presence of inorganic pyrophosphatase results in the formation of a highly fluorescentl-7AW–adenylate complex. Detection of this complex is based on its enhanced fluorescence at 315 nm excitation and 360 nm emission after the addition of ATP. This stereoselective reaction was used to develop an activity assay for TrpRS using commercially available racemicd,l-7AW. The assay can be used to determine the activity of TrpRS from samples which contain less than 1 nmol of enzyme in 250 μL of sample. Thus the enzyme activity can be assessed without resorting to a radioactive assay of tRNATrpacylation. A secondary use of the stereoselective assay was for confirming the presence of purel-7AW,d-7AW, or mixtures of the two enantiomers.d-7AW andl-7AW were prepared by reactingd,l-7AW with chloroacetic anhydride to formN-chloroacetyl-d,l-7AW (ClAc-7AW) followed by stereospecific proteolytic digestion of ClAc-7AW using carboxypeptidase A to produce the freel-7AW. Thel-7AW could be separated from unreactedN-chloroacetyl-7AW by reverse-phase HPLC. The TrpRS-based assay was able to unambiguously discriminate between the two enantiomers of 7AW. The assay was then used to identify which enantiomer of 7AW was present in resolved fractions of the tripeptidel-lysyl-d,l-7-azatryptophyl-l-lysine. Digestion of the resolved tripeptides with protease enzymes produced the freelordenantiomer of 7AW, which was easily identified using the TrpRS assay procedure.