We assume that mutations cause resistance by reducing the affinity of an antibiotic ligand for its target. Since we are only interested in whether a mutation increases or decreases the antibiotic's affinity for the target, the difference in binding free energy (DDG) between the wild type and mutant systems is calculated. This can be achieved by employing relative binding free energy (RBFE) methods, whereby a wild type amino acid is transmuted into the mutant along a nonphysical pathway defined by a progress coordinate, 0 ≤ l ≤ 1. For equilibrium-based methods, a series of short molecular dynamics (MD) simulations are performed at fixed values of l and the resulting DG values are related to the difference in binding free energy via a thermodynamic cycle (Fig. ). This approach has been shown to successfully predict if mutations in a relatively small protein, S. aureus DHFR (157 residues), confer resistance to trimethoprim, an antibiotic used to treat urinary-tract infections. In this paper we shall apply the same approach to two much larger protein complexes, the RNA polymerase (4,671 residues) and the DNA gyrase cleavage complex (1,473 residues), to assess how well we can predict the effect of seven and five mutations on the action of rifampicin and moxifloxacin, respectively.