In this manuscript, we first scrutinized the energy conservation of MD and AFE simulations across diverse systems employing varying timesteps, and examined their impact on the accuracy of AFE simulations where we employed a rigorous statistical t-test to assess the fidelity of binding free energies calculated utilizing a 4 fs timestep with SHAKE and HMR. In some cases, statistically significant derivations were observed in the derivative of the potential energy function with respect to the alchemical coupling parameter λ (i.e, dU dλ ) when utilizing a 4 fs timestep in comparison to a 0.5 fs timestep. This derivative is crucial for ascertaining alchemical free energy modifications through integration. These findings highlight the prospective implications of timestep selection on the accuracy of AFE simulations and underscore the imperative for meticulous consideration of this parameter.