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We conceptualize a novel approach towards enzyme assays based on the reversible and competitive binding of a fluorescent dye and the substrate as well as product of an enzymatic reaction to a macrocyclic host. This method was termed 'supramolecular tandem assay', and has been applied to inhibitor and activator screening, sensor array development, and enantiomeric excess determination of amino acids. The simple and rapid read-out by fluorescence allows their straightforward implementation into high-throughput screening.