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In 2010, the pan-assay interference compounds (PAINS) rule was proposed to identify false-positive compounds, especially frequent hitters (FHs), in biological screening campaigns, and has rapidly become an essential component in drug design. However, the specific mechanisms remain unknown, and the result validation and follow-up processing schemes are still unclear. In this review, a large benchmark collection of 600,000 compounds sourced from databases and the literature, including six common false-positive mechanisms, was used to ev