Here, our goal is to develop a stable MD-based protocol for the RBFE calculation (or the complex leg thereof, specifically; ∆G Complex,W ater A->B , Figure ), which can efficiently handle displacement and/or insertion of water(s) on modifying a ligand. Specifically, we seek to handle the case of a transformation between a ligand A and a ligand B in a protein's binding site, where ligand A binds with exactly one trapped water molecule and ligand B does not. Following the approach of Ge et al., we introduce an intermediate state in the alchemical transformation between the two ligands, where the intermediate state involves removal of the trapped water from the protein's binding site (Figure ). Our alchemical transformation, therefore, consists of three states: ligand A with the trapped water molecule (a favorable binding mode for ligand A), ligand A without the trapped water molecule (an unfavorable binding mode for ligand A), and ligand B without the trapped water molecule (favorable binding mode for ligand B). Ge et al. demonstrated their method by calculating the ABFEs of trapped water molecules (∆ G W ater , Figure ), i.e., calculating the binding affinities of water molecules to proteinligand complexes. Their calculations presented a proof of concept for free energy calculations for water displacement -in this case, the transformation from the system with ligand A and a trapped water molecule to the system of ligand A without the trapped water molecule. The calculations showed promise that the method could be expanded to run RBFE calculations between ligands that bind to the protein with different numbers of trapped waters. However, the protocol used in this prior work to perform these calculations was not without difficulties. For example, the authors encountered sampling problems with surrounding solvent water re-hydrating the emptied trapped water site and with the protein undergoing significant conformational transformations upon removal of the trapped water, ultimately influencing their free energy estimates.