An assay for enzymes which act on substrates to produce a single-stranded oligonucleotide product has been developed which involves the DNA polymerase-catalyzed extension of the oligonucleotide cleavage product using labeled nucleotides and a DNA template containing a 3' region complementary to the oligonucleotide product joined to a 5' region consisting of repeated nucleotide residues. The DNA polymerase extension assay does not involve gel electrophoretic separation and is amenable to high volume screening of potential inhibitors. Other key features of the assay are that it monitors the substrate cleavage reaction only at the correct position in the sequence, thereby discriminating against nonspecific cleavage products, and that it is sensitive enough to detect 200 attomoles of product.