The performance of the WaterKit method was evaluated using the MD simulations as a reference, analyzing 9 systems from the DUD-e dataset (fa10, fkb1a, hsp90α, ital, hivpr, fabp4, nram, kith and pgh2), plus the streptavidin-biotin complex, which water molecules have been extensively characterized and discussed in the literature . The WaterKit method was designed to facilitate the generation of trajectories similar to those obtained with MD simulations, so that they can similarly used as input for the Grid Inhomogeneous Solvation Theory (GIST) method. By sampling positions and orientations of individual water molecules, this approach can be used to calculate their individual thermodynamic properties, such as solute-water and water-water enthalpies (E sw , E ww , respectively), and translational and orientational entropies (TS trans , TS orient , respectively). MD simulations. To measure the variability among different simulations and obtain a robust reference, triplicates were performed for all the proteins, and compared to each other (Fig. ) By using progressively longer MD trajectories (from 100 to 30,000, corresponding to 0.1 ns to 30 ns), the GIST analysis shows that 30 ns is sufficient to achieve an adequate degree of convergence, except for proteins hivpr, nram, fabp4 and streptavidin (Fig. , S22, S16, S24). MD trajectories had to be extended to 100 ns (100,000 frames) for all triplicates of streptavidin, nram and hivpr, and to 200 ns (200,000 frames) for fabp4 protein. While satisfactory convergence was reached with 100 ns for the streptavidin protein, longer MD trajectories did not yield better convergence for nram, hivpr and fabp4 proteins (Fig. , S22, S16, S24). Despite system-specific convergence issues, the results showed that the different replicates were overall successful in identifying most of the common hydration sites across the different MD simulations, with a sensitivity of 0.65 and a precision of 0.83. Moreover, high correlation coefficients r 2 were obtained for each energy component, with values ranging from 0.75 for E ww to 0.88 for E sw . The average RMSD values observed are lower than 1 kcal/mol for E sw , E ww , and ∆G, and around 0.15 kcal/mol for TS trans and TS orient . Such difference of orders of magnitude between the component terms are in agreement with previous studies report-ing the predominance of enthalpy terms over entropy ones for water molecules, and consequently on their ∆G. In our results for MD simulations, observed values for the enthalpic terms span over a wider range of energy, about -20 kcal/mol for the most favorable hydration sites, compared to the entropic terms that did not exceed -3 kcal/mol. The largest estimated total entropic costs (TS trans + TS orient ) was around 6 kcal/mol, which is higher than the empirical upper bound of 2.1 kcal/mol at 300K proposed by Dunitz 126 for transferring a water molecule from the bulk to a binding site. This is likely a consequence of the protein flexibility restraints imposed by the GIST protocol, resulting in limited degrees of freedom for water.