SUMMARY: Evaluation of the capacity for separating actives from challenging decoys is a crucial metric of performance related to molecular docking or a virtual screening workflow. The Directory of Useful Decoys (DUD) and its enhanced version (DUD-E) provide a benchmark for molecular docking, although they only contain a limited set of decoys for limited targets. DecoyFinder was released to compensate the limitations of DUD or DUD-E for building target-specific decoy sets. However, desirable query template design, generation of multiple decoy sets of similar quality, and computational speed remain bottlenecks, particularly when the numbers of queried actives and retrieved decoys increases to hundreds or more. Here, we developed a program suite called RApid DEcoy Retriever (RADER) to facilitate the decoy-based assessment of virtual screening. This program adopts a novel database-management regime that supports rapid and large-scale retrieval of decoys, enables high portability of databases, and provides multifaceted options for designing initial query templates from a large number of active ligands and generating subtle decoy sets. RADER provides two operational modes: as a command-line tool and on a web server. Validation of the performance and efficiency of RADER was also conducted and is described. AVAILABILITY AND IMPLEMENTATION: RADER web server and a local version are freely available at http://rcidm.org/rader/ . CONTACT: [email protected] or [email protected] . SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.