We used fluorogenic substrates, namely, alkyldiacyl glycerols labeled with a fluorophore and a fluorescence quencher, to measure lipase activities. For this optical lipase assay it is necessary that the water-insoluble fluorogenic substrates are dispersed in an aqueous medium in the presence of appropriate detergents in order to obtain well-defined and reproducible systems with high optical transparency. The activity of lipases critically depends on the supramolecular organization of the substrate. Therefore, we tested different solutions containing nonionic surfactants and organic phases for their potential use as host systems for fluorogenic lipids. We then determined the pseudoternary phase diagram for the system buffer-alkyl polyglucoside-hexanol, which showed the highest apparent enzyme activities, in the buffer corner. We performed structural investigations on micellar sugar surfactant solutions within the L-1 phase with and without hexanol using small-angle neutron scattering (SANS), small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) in order to investigate a possible correlation between Lipase activity and the structure of the aggregates present in the host system.