The invention relates to determination of the profile of unbound free fatty acids (FFAu) in biological samples, such as human and animal blood specimens and plant and animal oils, by measuring the fluorescence response of sets of different fluorescently labeled fatty acid binding proteins (probes) that undergo a change in fluorescence ratio at 2 wavelengths upon binding an FFAu. Use of these profiles in human and animal disease, in basic research, in drug development and in industrial uses is disclosed.