A method for determining the relative concentrations of unbound free fatty acid (FFAu) in a sample comprising the steps of: (a) providing a set of one or more FFAu specific probes, wherein the set of one or more FFAu specific probes comprises probes with complementary specificities for the FFAu of interest and/or the FFAu in the sample and wherein the probes are intracellular lipid binding proteins (iLBPs) and are fluorescently labeled at a cysteine or lysine residue; (b) combining each probe with a separate aliquot of the sample; (c) measuring a fluorescence index at two different emission wavelengths for each probe in the combined probe and aliquot of the sample; (d) calculating the ratio of the fluorescence index at the two wavelengths for each probe in the sample; (e) expressing each probe's ratio in terms of sum of contributions from the different FFAu to which the probes respond; and (f) solving a set of ratio equations for the relative concentration of the FFAu concentrations; R j − R o j = ∑ i = N FFA i Q i j Kd i j Rm i j − R j wherein j is the probe; wherein FFAi is an individual FFAu; for each probe j, wherein R j is the blank subtracted fluorescence intensity at wavelength 1 divided by the blank subtracted fluorescence intensity at wavelength 2 in the presence of each FFAu from 1 to N, wherein N is the total number of FFAu; wherein R O j is the blank subtracted fluorescence intensity at wavelength 1 divided by the blank subtracted fluorescence intensity at wavelength 2 in the absence of each FFAu for each probe; wherein Rm j i is the R value at saturation; wherein Kd j i is the equilibrium dissociation constant; wherein Q j i is the ratio of the intensity at one of the two wavelengths with no FFAu bound to the probe at saturation; wherein more than one probe is used and the number of probes is greater than the number of FFAu to be solved for and for which the relative concentrations of FFAus are determined using a fitting method. (a) providing a set of one or more FFAu specific probes, wherein the set of one or more FFAu specific probes comprises probes with complementary specificities for the FFAu of interest and/or the FFAu in the sample and wherein the probes are intracellular lipid binding proteins (iLBPs) and are fluorescently labeled at a cysteine or lysine residue; (b) combining each probe with a separate aliquot of the sample; (c) measuring a fluorescence index at two different emission wavelengths for each probe in the combined probe and aliquot of the sample; (d) calculating the ratio of the fluorescence index at the two wavelengths for each probe in the sample; (e) expressing each probe's ratio in terms of sum of contributions from the different FFAu to which the probes respond; and (f) solving a set of ratio equations for the relative concentration of the FFAu concentrations; R j − R o j = ∑ i = N FFA i Q i j Kd i j Rm i j − R j wherein j is the probe; wherein FFAi is an individual FFAu; for each probe j, wherein R j is the blank subtracted fluorescence intensity at wavelength 1 divided by the blank subtracted fluorescence intensity at wavelength 2 in the presence of each FFAu from 1 to N, wherein N is the total number of FFAu; wherein R O j is the blank subtracted fluorescence intensity at wavelength 1 divided by the blank subtracted fluorescence intensity at wavelength 2 in the absence of each FFAu for each probe; wherein Rm j i is the R value at saturation; wherein Kd j i is the equilibrium dissociation constant; wherein Q j i is the ratio of the intensity at one of the two wavelengths with no FFAu bound to the probe at saturation; wherein more than one probe is used and the number of probes is greater than the number of FFAu to be solved for and for which the relative concentrations of FFAus are determined using a fitting method.