In this paper we introduce a generalized and efficient Separated Topologies approach and show that it works on pharmaceutically relevant systems. We developed a stable implementation of the approach and provide a package to setup input files for running SepTop in Gromacs. For the orientational restraints applied in the SepTop approach we wrote an algorithm that picks atoms for restraints automatically based on heuristics we developed here to select relatively stable atoms in the ligand and protein (Section 2.2). This selection algorithm avoids several pitfalls and provides a robust and fairly general solution, at least in the systems investigated herein. We also developed a general and efficient protocol for running SepTop RBFE with a reasonable λ schedule that worked on a variety of targets and we implemented an ϵ-HREX protocol for enhanced sampling. 6.1. SepTop performed well on the test systems in this study. We tested the method on several diverse, pharmaceutically relevant systems and reported performance and any resulting insights into strengths, weaknesses and challenges. We first validated the method on a system with standard RBFE results available (TYK2) and obtained comparable results to a standard RBFE method as well as achieved good convergence. We then applied the method to scaffold hopping transformations in the ERα system such as ring breaking transformations and compared results to other RBFE methods that allow scaffold hopping transformations. Here, we found that the preparation of the protein, and more specifically modeling of missing loops and the protonation state of histidine residues, had an impact on the binding free energy, highlighting both the importance of careful protein preparation as well as the challenge to identify whether differences in results between different RBFE methods originate from the methods itself or differences in the input structures. After validating the method on these smaller datasets, we tested SepTop on two larger datasets, MALT1 and BACE1, with transformations not as straightforward to run with typical RBFE. For both targets, results correlated well with experiment and good error statistics were achieved. We analyzed cycle closures as a convergence check and identified sampling problems due to slow rotamer sampling of different dihedral angles in the ligand.