Fluorescence resonance energy transfer (FRET) systems in which a peptide sequence contains both a fluorophore and an internal quencher are amongst the best methods available for protease analysis and characterization. Numerous proteases have been studied using this method, including trypsin which cleaves the FRET-based system Dabcyl-Gly-Pro-Ala-Lys-Leu-Ala-Ile-Gly-Edans, cathepsin B which cleaves Abz-Lys-Leu-X-Phe-Ser-Lys-Gln-EDDnp (where X is Cys(SBz), Thr(OBz), or Ser(OBz) and EDDnp is 2,4-dinitrophenylethylenediamine), leukotriene D 4 hydrolase which cleaves e-DNP-l-Lys-D-Amp (where Amp is 2-amino-3-(7-methoxy-4-coumaryl)propanoic acid) and caspase 1 and 3 which cleave the FRET systems BFP-Tyr-Val-Ala-Asp-GFP and BFP-Asp-Glu-Val-Asp-GFP, respectively (where BFP is blue fluorescent protein and GFP is green fluorescent protein) [4] to name but a few. A host of methods have been developed using this methodology to determine the most appropriate substrate for a particular protease. For example, Meldal et al. have reported a powerful general method for the use of a splitand-mix approach to libraries of FRET-based peptides for onbead screening to determine optimal substrates for a range of proteases including subtilisin carlsberg, [5] FRET-based hexapeptide libraries have been used to map the S'-subsite specificity of serine proteases using solution-based assays, [6] and a positional scanning substrate library has been used to determine the specificity of ICE (ΒΌ interleukin-converting enzyme). [7] In this paper a new method for the detection of proteolytic activity is presented using internal fluorescence quenching between fluorophores of the same type. These are attached to a dendrimer-type structure, thereby giving a high local concentration of fluorophore needed for quenching yet eliminating the need for a separate quenching moiety. This method simplifies the synthesis of substrates for assay compared to traditional FRET, while being very sensitive due to the amplification nature of the assay as multiple COMMUNICATIONS