Protein stability and unfolding are central to understanding structure-function relationships, enzyme engineering, misfolding diseases, and drug discovery. This Topical Collection curates state-of-the-art experimental and computational methodologies that quantitatively probe protein stability landscapes and unfolding mechanisms under diverse perturbations, including chemical denaturants, temperature, pH, and ligand binding. The collection emphasizes integrative approaches combining biophysical experiments with molecular modeling, featuring chemical denaturation assays, thermal unfolding, circular dichroism, fluorescence spectroscopy, differential scanning calorimetry, hydrogen-deuterium exchange, NMR, cryo-EM, molecular dynamics simulations, enhanced sampling methods, and stability prediction algorithms. A key objective is to standardize reproducible, visualized protocols that lower the barrier for researchers entering the field, with particular focus on mechanistic dissection of denaturant-induced unfolding, engineering thermostable proteins, and linking stability perturbations to biological function and disease.