Fluorescence resonance energy transfer (FRET) peptide substrates are often utilized for protease activity assays. This study has examined the preparation of FRET triple-helical peptide (THP) substrates using 5-carboxyfluorescein (5-Fam) as the fluorophore and 4,4-dimethylamino-azobenzene-4'-carboxylic acid (Dabcyl) as the quencher. The Nα-(9-fluorenylmethoxycarbonyl)-Nε-(5-carboxyfluorescein)-l-lysine [Fmoc-Lys(5-Fam)] building block was synthesized utilizing two distinct synthetic routes. The first involved copper complexation of Lys while the second utilized Fmoc-Lys with microwave irradiation. Both approaches allowed convenient production of a very pure final product at a reasonable cost. Fmoc-Lys(5-Fam) and Fmoc-Lys(Dabcyl) were incorporated into the sequence of a THP substrate utilizing automated solid-phase peptide synthesis protocols. A second substrate was assembled where (7-methoxycoumarin-4-yl)-acetyl (Mca) was the fluorophore and 2,4-dinitrophenyl (Dnp) was the quencher. Circular dichroism spectroscopy was used to determine the influence of the fluorophore/quencher pair on the stability of the triple-helix. The activity of the two substrates was examined with three matrix metalloproteinases (MMPs), MMP-1, MMP-13, and MT1-MMP. The combination of 5-Fam as fluorophore and Dabcyl as quencher resulted in a triple-helical substrate that, compared with the fluorophore/quencher pair of Mca/Dnp, had a slightly destabilized triple-helix but was hydrolyzed more rapidly by MMP-1 and MMP-13 and had greater sensitivity. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 347–355, 2013.