Here, we perform a systematic investigation of the effect of ensemble size on alchemical ABFE predictions by varying it from 1 to 100 and determine an appropriate value for optimal performance, that enables achieving chemical accuracy (< 1 kcal/mol) and desired precision (≤ 0.5 kcal/mol). Further, we point Table : Overview of computational requirements for EQ and NEQ methods of ABFE calculations. The number of λ-intermediates in case of EQ calculations is taken to be 15 for the complex leg and 13 for the ligand leg. In the case of NEQ, the computational requirements for ∆G com restr are taken equal to those for ∆G com alch for comparison. Although in this study, ∆G com restr from the EQ protocol (calculated using 12 λ-intermediates) has been reused in the case of NEQ. out that it should be considered a flexible parameter with adjustments made whenever necessary. Based on this analysis, we determine that an ensemble size of 10 is the most appropriate for alchemical ABFE simulations in the protein environment and use this value as a standard in all our calculations. However, for the ligand decoupling in water, an ensemble size of 5 is found sufficient due to lesser fluctuations in energy derivatives across replicas as also shown in our previous study . It is noteworthy that larger ensembles are required for a thorough investigation of non-normal distributions.