Dynamic and steady-state fluorescence spectroscopic properties of a dye probe measured in a multicomponent biological system are often required to be separated into the spectra and lifetimes of individual spectroscopically distinct species. The conventional method of obtaining decay-associated spectra fails when the lifetimes of the fluorophore in the membrane phase and in the aqueous phase are very close to each other. This paper describes a global analysis method which takes advantage of the known spectrum and lifetime of the dye in the aqueous phase. This method is used to identify the spectra for two fluorescent species (lifetimes, 0.84 and 1.97 ns) of the dye DODCI in EggPC vesicle membranes by keeping the spectrum and lifetime (0.68 ns) of the dye in the aqueous phase as fixed parameters. The structural identity of the two membrane-bound dye species was established by the effect of refractive index and/or viscosity of the aqueous medium on the lifetimes.