Stability is one of the most important characteristics of proteins employed as biocatalysts, biotherapeutics, and biomaterials, and the role of computational approaches in modifying protein stability is rapidly expanding. We have recently identified stabilizing mutations in haloalkane dehalogenase DhaA using phylogenetic analysis but were not able to reproduce the effects of these mutations using force-field calculations. Here we tested four different hypotheses to explain the molecular basis of stabilization using structural, biochemical, biophysical, and computational analyses. We demonstrate that stabilization of DhaA by the mutations identified using the phylogenetic analysis is driven by both entropy and enthalpy contributions, in contrast to primarily enthalpy-driven stabilization by mutations designed by the force-field Comprehensive bioinformatics analysis revealed that more than half (53%) of 1 099 evolution-based stabilizing mutations would be evaluated as destabilizing by force-field calculations. Thermodynamic integration considers both folded and unfolded states and can describe the entropic component of stabilization, yet it is not suitable for predictive purposes due to its high computational demands. Altogether, our results strongly suggest that energetic calculations should be complemented by a phylogenetic analysis in protein-stabilization endeavors.