The non-covalent binding free energy contribution of the inhibitors to the M pro target was computed using alchemical absolute binding free energy calculations. Alchemical ABFE calculations provide a direct of computing ligand binding energies within a rigorous statistical thermodynamic framework. This approach employs a nonphysical thermodynamic cycle to compute inhibitor binding free energies and considers the conformational entropy and dynamics of both the protein target and inhibitor upon binding. ABFE calculations can yield highly accurate binding affinities for ligands that agree very well with experiment. Recently, we employed this approach to estimate the binding affinity of the peptidomimetic inhibitors N3 and α-ketoamide of the SARS-CoV-2 M pro . In this work, we calculated the absolute binding free energies of the inhibitors to the M pro target using both non-enhanced and enhanced sampling techniques within the equilibrium ABFE framework. For the enhanced ABFE simulations, a Hamiltonian replica -exchange molecular dynamics (H-REMD) algorithm 34 was employed to facilitate sampling convergence in the free energy estimates. Table provides a summary of the binding free energy results with and without H-REMD for the complexes studied in this work.