Unfortunately, the probabilities  and  cannot be measured directly.  Instead, we assay the relative population of folded molecules using various structural probes, e.g., absorbance at 287 nm (which reports on the solvent exposure of tryptophan and tyrosine), far-ultraviolet circular dichroism (180-250 nm, which reports on the secondary structure of the protein backbone), dual polarisation interferometry (which reports the molecular size and fold density) and near-ultraviolet fluorescence (which reports on changes in the environment of tryptophan and tyrosine).  However, nearly any probe of folded structure will work; since the measurement is taken at equilibrium, there is no need for high time resolution.  Thus, measurements can be made of NMR chemical shifts, intrinsic viscosity, solvent exposure (chemical reactivity) of side chains such as cysteine, backbone exposure to proteases, and various hydrodynamic measurements.
To convert these observations into the probabilities  and , one generally assumes that the observable  adopts one of two values,  or , corresponding to the native or unfolded state, respectively.  Hence, the observed value equals the linear sum
By fitting the observations of  under various solution conditions to this functional form, one can estimate  and , as well as the parameters of .  The fitting variables  and  are sometimes allowed to vary linearly with the solution conditions, e.g., temperature or denaturant concentration, when the asymptotes of  are observed to vary linearly under strongly folding or strongly unfolding conditions.