A new methodology for the detection of lipid flip was developed. This methodology relies on the quenching of the fluorescence of the cascade-blue-labeled lipid through complex formation with a membrane-impermeable cyclen-tetranaphthalenethiourea synthetic receptor for this dye. The high affinity of the receptor to cascade-blue label allows the use of micromolar concentrations of this receptor during the experiment. At these low concentrations, the receptor does not interfere with the membrane integrity and, therefore, renders this new methodology less invasive to the model and cell membranes than commonly utilized 7-nitro-1,2,3-benzoxadiazol-4-yl (NBD)-dithionite methodology. Unlike with the NBD-dithionite assay, where the fluorescence quenching of the NBD group is achieved through its chemical modification, this new assay relies on the noncovalent interactions between cascade-blue label and the receptor. Therefore, the quenching can be reverted by either competitive displacement of the lipid-attached label with a water-soluble substrate or by enzymatic degradation of the receptor leading to the label release and fluorescence dequenching. We demonstrate that this new methodology is suitable for the study of lipid flip in both model spherical bilayer membranes and in-vitro experiments.