The aqueous phase monomers of fatty acids (FFA) appear in many steps of fat metabolism. Understanding metabolism requires that accurate measurements of FFA levels be determined in enzyme-mediated as well as in membrane and protein binding reactions. Measuring long chain FFA levels with sufficient sensitivity and temporal resolution is now possible using fluorescent probes constructed by ligating fluorescent groups and fatty acid binding proteins. In this paper we provide a practical description of the use of ADIFAB, the acrylodan labeled intestinal fatty acid binding protein. We describe with specific examples how ADIFAB can be used to determine, (1) FFA concentrations in aqueous solutions, (2) binding affinities of fatty acid binding proteins, (3) membrane/water partition coefficients, (4) lipase activities, and (5) serum levels of FFA.