Abstract: The present invention is generally directed to biological assays. More specifically it is directed to FRET-based assays using particularly effecting FRET pairs, methods for performing such assays and the molecules utilized in the assays. In a composition aspect, the present invention is directed to a FRET-based protease substrate selected from the list of substrates shown in the Detailed Description section above. In a method aspect, the present invention is directed to a method of performing a FRET-based assay, where the assay includes the following steps: adding a solution or suspension containing one or more different proteins to a reaction vessel; adding a liquid comprising a FRET-substrate to the reaction vessel, wherein the FRET-substrate is selected from the list of substrates shown in the Detailed Description section above. In a kit aspect, the present invention is directed to a kit for performing a FRET-based assay, wherein the kit comprises a protease substrate.