Free energy differences in biomolecular systems are often required to quantitatively characterize the structure and dynamics of biomolecular interactions underlying multiple biological and biochemical processes. One such important quantity is the binding free energy (BFE) of a small molecule ligand to a protein. BFEs can be categorized into two broad classes: absolute BFE (ABFE) and relative BFE (RBFE). ABFE is the difference in free energy between the protein-ligand complex and the free protein and ligand in solution, referred to as the standard binding free energy. Δ𝐺 𝑏𝑖𝑛𝑑 𝑜 This quantity can be measured experimentally, e.g., by surface plasmon resonance or isothermal titration calorimetry. ∆𝐺 𝑏𝑖𝑛𝑑 ° is related to the ligand dissociation constant Kd by the following relation: