Free energy calculations of protein ligand binding are of great interest to medicinal chemists, because they can be used to guide structure-based drug design. Among the existing computational techniques, molecular dynamics-based alchemical free energy calculations are unique in their accuracy and solid theoretical basis, and therefore show great potential for a widespread use in pharmaceutical drug discovery. Recently, we adopted alchemical relative free energy calculations for evaluating and predicting binding affinities of a series of serine protease factor Xa inhibitors, which plays an important role in the blood coagulation cascade. Using a combination of free energy calculations and experimental validation, a large perturbation in the binding affinity of several new fXa ligands could be identified. Moreover, analysis and comparison of inhibitors with different substituents and scaffolds from the calculations lead us to a better understanding of the underlying interactions between the inhibitors and their target.