Chemical and thermal denaturation methods have been widely used to investigate folding processes of proteins in vitro. However, a molecular understanding of the relationship between these two perturbation methods is lacking. Here, we combined computational and experimental approaches to investigate denaturing effects on three structurally different proteins. We derived a linear relationship between thermal denaturation at temperature T(b) and chemical denaturation at another temperature T(u) using the stability change of a protein (ΔG). For this, we related the dependence of ΔG on temperature, in the Gibbs-Helmholtz equation, to that of ΔG on urea concentration in the linear extrapolation method, assuming that there is a temperature pair from the urea (T(u)) and the aqueous (T(b)) ensembles that produces the same protein structures. We tested this relationship on apoazurin, cytochrome c, and apoflavodoxin using coarse-grained molecular simulations. We found a linear correlation between the temperature for a particular structural ensemble in the absence of urea, T(b), and the temperature of the same structural ensemble at a specific urea concentration, T(u). The in silico results agreed with in vitro far-UV circular dichroism data on apoazurin and cytochrome c. We conclude that chemical and thermal unfolding processes correlate in terms of thermodynamics and structural ensembles at most conditions; however, deviations were found at high concentrations of denaturant.