We employ the double decoupling scheme using the thermodynamic cycle shown in Figure . The physical process of binding is decomposed into several alchemical steps with a free energy value (∆G) associated with each step. The binding affinity of the ligand-protein complex (∆G b ) is given by the sum of these individual ∆G values. The process begins with the decoupling of the ligand in solvent such that the electrostatic and van der Waals interactions between ligand and solvent are turned off (-∆G lig alch ). This is followed by restraining the decoupled ligand within the binding site of protein using a set of restraints proposed by Boresch et al. (∆G lig restr + ∆G prot conf ). It should be noted here that we add an additional free energy term in this step (∆G prot conf ) that captures the change in protein conformation from apo state to holo state. This term is usually ignored and leads to systematic errors in The process of binding is divided into a series of nonphysical transformations each with an associated ∆G: ligand decoupling from solvent (-∆G lig alch ), restraining the decoupled ligand within the binding pocket of protein (∆G lig restr + ∆G prot conf ) and ligand coupling in the protein environment (∆G com alch ) while relaxing the restraints (-∆G com alch ). The binding free energy (∆G b ) is given by the sum of all these terms. Ensemble sizes need to be varied for various steps in this cycle to keep errors under control. the predicted ABFEs. Finally, the ligand is coupled with the protein environment by turning on ligandprotein electrostatic and van der Waals interactions while relaxing the restraints (∆G com alch -∆G com restr ). The final two steps of coupling the ligand and removing its restraints may be performed either seperately (coupling followed by relaxation) or simultaneously. We show that both yield almost identical results while the latter reduces the computational costs substantially (see Figure and the related section in the Supporting Information for details). The terms ∆G com alch , -∆G lig alch and -∆G com restr are obtained using simulations, whereas ∆G lig restr is calculated analytically as proposed by Boresch et al. . However, it is not straightforward to obtain ∆G prot conf ; we propose a way to estimate it as discussed in the Results section. We compare two distinct approaches -equilibrium (EQ) and non-equilibrium (NEQ) -as described in sections 2.2 and 2.3. The above thermodynamic cycle is used with both these approaches and the binding affinity is given by the following equation: