We evaluated ABFE as a virtual screening tool based on data for the protein targets BACE1, CDK2, and thrombin. For each target, we docked and scored a mixture of several hundred known actives and 18,000-28,000 presumed inactives (decoys) drawn from the DUD-E resource. We then compared the ability of docking and ABFE to distinguish actives from decoys among tractable sets of compounds with the very best docking scores (Tier 1 compounds) and among compounds with somewhat worse docking scores (Tier 2 compounds). In order to put these results into context, we also compared the accuracy of our docking calculations across all compounds with those previously reported in the original DUD-E paper . This section presents these ABFE and docking results and evaluates factors that limit ABFE precision and accuracy and how these factors suggest future directions to improve the ABFE approach.