The concentration of long-chain (14–18 carbons) fatty acids (FA) free in solution (unbound) is difficult to measure directly because of the low aqueous solubility of these common dietary FA. One indirect and convenient way to measure the concentration of unbound FA is a method using the fluorescent-(acrylodan) labeled intestinal FA-binding protein (ADIFAB). Under appropriate conditions, ADIFAB fluorescence measures unbound FA, regardless of any third phase such as albumin, FA-binding proteins, or membranes. With knowledge of the total amount of FA in the system and the assumption that the amount of FA bound to ADIFAB is negligible, equilibrium constants or partition coefficients for FA in equilibrium with the third phase can be calculated. Herein, the use of ADIFAB is described to measure unbound FA concentration using oleic acid as a typical long-chain FA. Attempts were not made to calibrate the accuracy of ADIFAB for FA concentration, but to investigate its reliability and reproducibility under differing buffer conditions. It is shown that ADIFAB fluorescence is sensitive to biologically prevalent ions and that calibration curves must be constructed for conditions that do not closely match those previously published. The results with in vitro systems suggest that there will be caveats with the application of ADIFAB to measure FA concentrations in vivo, where the precise environment of the probe is not known or cannot be tightly controlled.