We are often interested in studying the effects of small structural changes on the binding affinity of a series of structurally similar drug-like molecules. Experimentally, this is often done in structure-activity relationship (SAR) studies, while computationally we can use molecular dynamics (MD) methods. Simulating binding events directly with unbiased MD requires the observation of multiple binding events for sufficient statistics and due to the timescales of MD, more efficient methods are needed. One class of such methods is relative binding free energy (RBFE) calculations. These make use of a thermodynamic cycle to evaluate a relative free energy of binding ∆∆G bind , by simulating an unphysical path of an alchemical transformation of one molecule into another molecule. By computing this transformation in a water box (unbound ), and while bound to the protein, a relative free energy of binding can be computed for a given ligand pair L 1 and L 2 .