The so-called "relative" binding free energy (RBFE) approach calculates the difference in potency between two similar ligands. During the simulations, one ligand is converted into the other by alchemical transformations of the atoms that vary between the two ligands. Common atoms from one ligand are mapped on top of those from the other ligand, resulting in either a single set of coordinates of the two ligands in the end states (single topology) or a single set of coordinates for the common substructure while presenting atoms that differ between the two ligands separately (hybrid topology). This approach is less computationally demanding and has lower statistical uncertainties than absolute binding free energy calculations (ABFE). It also has advantages, e.g. if both ligands introduce similar conformational changes in the protein, such slow motions do not have to be sampled since the binding site is never empty. This RBFE approach is most suitable for comparing binding of related ligands and is routinely applied in drug discovery. The RBFE approach, however, essentially re-quires that the two ligands share a common scaffold which can be preserved (allowing the ligand to retain its binding mode) while modifying atoms which are not retained. This requirement for a common scaffold provides a critical challenge, especially in early-stage drug discovery where complex modifications to ligands are common. This limitation prevents these techniques from being as useful as they could be in guiding drug design. Scaffold hopping approaches allow for larger transformations, for example ring opening and ring size change transformations; however, the transformation size is limited, since ligands still need to share a common core. Moreover, in common practice RBFE calculations need ligands to have a shared binding pose and/or protein conformation. Additionally, RBFE calculations require atom mapping and the construction of the "dummy atoms" must be done carefully to ensure that the energy contribution of the decoupled dummy atoms cancels out between the complex and the solvent legs of the thermodynamic cycle. For example, if dummy atoms are connected to the rest of the system by more than one bond, the energy contribution does not cancel out automatically. Additionally, angle and torsional terms can introduce considerable complexity if not handled with great care. This concern does not apply to systems where all ligand atoms are transformed to dummy atoms, such as in ABFE. Alternatively, the "absolute" approach computes the potency of individual ligands directly, usually through a thermodynamic cycle where a ligand is decoupled in the binding site -meaning all its non-bonded interactions are turned off -and coupled in the solvent where the interactions are turned back on. Since ligands are treated individually, they do not need to share a common scaffold and can be structurally diverse. This means that ABFE could be used even in early project stages where structurally diverse ligands are common, and has been proposed to serve as a final scoring stage in virtual high throughput screening before selecting molecules for experimental testing. Recent studies showed that ABFE can achieve a good correlation between predicted and experimen-tal binding free energies across different systems, and even can be used to estimate binding to different proteins, allowing computation of the selectivity of ligands for a particular target. However, a major limitation of the ABFE approach is that it can produce larger statistical uncertainties in the predicted potency of the ligand compared to relative approaches (see below), especially in systems where the target undergoes larger conformational changes upon ligand binding. For example, consider a protein undergoing a slow flap-closing motion upon ligand binding, such as HIV protease; an ABFE calculation would need to sample the unbound state to correctly compute the true binding free energy. Such protein motions are not sampled on the typical timescale of MD simulations, resulting in inaccurate potency predictions. Slow degrees of freedom require long sampling times or the use of enhanced sampling techniques, which can increase computational costs. As a result, the ABFE method is not routinely applied in drug discovery projects. If, when the ligand is decoupled, all structures are metastable in something like the bound state one can obtain relative results from ABFE without having to sample apo-holo protein conformational transitions. However, this is not always the case, as discussed in Section 6.