To assess the impact of the timestep selection on Alchemical Free Energy (AFE) simulations, we first evaluated the energy conservation with different timesteps and then the reproducibility of dU dλ , to be explained below. To cover different situations of simulations, we chose two simple transforming molecular systems, cyclic adenosine monophosphate (cAMP) to cyclic adenosine-3 ′ ,5 ′ monophosphorothioate (Rp-cAMPS) , ethane to ethanol, and One protein- Thermodynamic Integration (TI) method AFE simulations are frequently employed within the domain to ascertain binding-free energies with a commendable degree of accuracy. A method that is extensively adopted for the computation of AFE is Thermodynamic Integration (TI) which demonstrates particular efficacy in quantifying the impacts of alchemical transformations. Here we will start with a brief description of the TI method.