Sirtuins are nicotinamide dinucleotide-dependent class III histone deacetylases catalyzing various physiological processes involved in cell proliferation, differentiation, apoptosis, and ageing. This makes them attractive targets in drug research. In order to simplify sirtuin substrates for assay development, two N(ɛ)-acetyllysine derivatives, N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine amide, and N(ɛ)-acetyl-N(α)-(4-methyl-7-methoxycoumarin)lysine methyl ester were synthesized and evaluated as substrates for human SIRT1 in a capillary electrophoresis-based enzyme assay. Substrate, deacetylated product, and the coproduct nicotinamide were separated in a 200 mM phosphate/Tris buffer at pH 2.85. Field-amplified sample injection was employed to achieve sufficient assay sensitivity. While the ester derivative was not recognized by the enzyme, the amide substrate was effectively converted to the deacetylated product. The assay was subsequently validated with respect to range, linearity, limit of detection, and limit of quantification. Michaelis-Menten kinetic parameters, K(m) = 83 μM and V(max) = 6.8 μM/min were determined. The applicability of the assay for inhibitor screening was demonstrated using the known inhibitors sirtinol and the suramin derivate NF258. Resveratrol did not increase the deacetylation rate at concentrations of up to 200 μM. Docking experiments revealed the necessity of an amide function at the C-terminus of nonpeptide substrates while more structural freedom is tolerated at the N-terminus of N(ɛ) -acetyllysine.