Some recent studies have addressed the challenge of sampling the trapped waters in the course of ligand transformations by inserting or deleting waters in the binding site using enhanced sampling methods, simultaneously with the ligand transformation. These methods normally require long simulation times to converge, and they can result in differences in RBFEs when the simulations are started with or without the known trapped waters. Such differences in the RBFEs are known as hysteresis. Recent studies on accessing the accuracies and efficiencies of these enhanced sampling methods in rehydrating trapped water sites showed that for several systems, the simulation timescale necessary to accurately predict the locations of the trapped waters might be well beyond that of typical free energy calculations. Alternatively, if the positions and numbers of trapped waters are known for two ligands a priori, one might seek an efficient method to calculate the RBFE of the ligands with each in the context of its respective water structure or network, without relying on fully sampling potential water structures with each ligand present, i.e., without using the insertion and deletion attempts mentioned above to determine and sample the correct water structure, which is already known. Essentially, the choice may be between a combined approach of sampling all potentially relevant water structures along with the ligand transformation, i.e. mixing a challenging problem of the trapped waters' sampling with a ligand RBFE calculation, or a separated approach of determining the respective positions of the trapped waters for each ligand first, and then holding them essentially fixed during the ligand RBFE calculation. ), the two components of the RBFE calculation. For the complex leg of the RBFE calculation, we use the separation of states approach where we first introduce a new state of the system (bottom) with a binding mode of the ligand A without the trapped water. In this state of the system, the trapped water is displaced from the binding site of the protein into the bulk solvent. From this binding mode of ligand A without the trapped water, we transform the ligand A into another ligand B that is known to displace the trapped water when it binds.