The authors’ laboratory has applied a series of different fluorescence assays for monitoring the binding and transport of fatty acids (FA) in model and biological membranes. The authors recently expanded their fluorescent assays for monitoring the adsorption of FA to membranes to a total of three probes that measure different aspects of FA binding: (1) an acrylodan-labeled FA-binding protein, which measures the partitioning of FA between membranes and the aqueous buffer; (2) the naturally occurring fluorescent cis -parinaric acid, which specifically measures the insertion of the FA acyl chain into the hydrophobic core of the phospholipid bilayer, and (3) a fluorescein-labeled phospholipid (N-fluorescein-5-thiocarbomoyl-1,2,dihexadecanoyl-sn -glycero-3-phosphoethanolamine), which specifically measures the arrival of the FA carboxyl at the outer leaflet of the membrane. None of these probes allow the transmembrane movement of FA to the inner leaflet to be measured. FA translocation (flip-flop) is typically measured directly, using a pH-sensitive fluorophore such as 8-hydroxypyrene-1.3.6-trisulfonic acid or 2′,7′-bis -(2-carboxyethyl)-5-(and-6)- carboxyfluorescein. These probes detect the release of protons from unionized FA that have diffused through the membrane to the inner leaflet. Because adsorption of FA to the outer leaflet must occur before flip-flop, these probes measure the effects of the combined steps of adsorption and translocation. In this chapter, detailed methods are provided on how to monitor the transport of FA through protein-free model membranes, and some of the fluorescent artifacts that may arise with the use of these probes are addressed. Also, experiments designed to investigate such artifacts, and improve the reliability and interpretation of the data are described.