Aberrant regulation of kinase and phosphatase activities is implicated in various diseases, including cancer, diabetes, and inflammation. Thus, high-throughput screening (HTS) has become a focused strategy for the identification of kinase and phosphatase inhibitors. With a growing number of these enzymes becoming available for HTS, rapid identification of substrates has become pertinent. Several substrate panel screening assays exist that allow the researcher to test dye-labeled peptides for kinase or phosphatase activity. Here we introduce a method that uses readily available biotinylated peptides instead of dye-labeled substrates, which are costly and limited in availability. After enzymatic phosphorylation, biotinylated peptides are coupled to streptavidin-quencher conjugates, which then associate with a fluorescent polymer via phosphate-metal ion interaction between the reacted biotinylated peptide complex and the polymer. As a result, quencher and polymer are brought into a proximity that allows electron transfer from the polymer to the dye. The Dylight(647) (Pierce, Rockford, IL) dye was identified as an efficient electron transfer molecule that allows assays to be monitored using two emission wavelengths simultaneously, 490 nm from the polymer and 685 nm from the transferred emission of the dye. Assays are homogeneous and show comparable sensitivities to assays performed with direct-labeled dyes. When applied to a limited screen using previously characterized peptides, substrates for two kinases and one phosphatase were correctly identified. Further, ratiometric analysis of polymer quenching and transferred emission accurately detected inhibitors in a compound screen against protein kinase A, protein kinase Calpha, and protein tyrosine phosphatase 1B with limited interferences from colored compounds and with Z factors of >0.7.