biostudies-literatureTsai CNBoris Family Fund for Health Research ExcellenceCanadian Institutes of Health ResearchCIHR100057https://www.ebi.ac.uk/biostudies/studies/S-EPMC7580248biostudies-literatureUnknown1(2)Anti-virulence therapies are under active investigation as antibiotic alternatives; however, their identification from large-scale chemical libraries poses a unique challenge. The dispensability of virulence factors for growth <i>in vitro</i> precludes conventional, optical density-based screening methods. Here, we provide a protocol for high-throughput screening with a cell-based, promoter reporter platform. We describe the use of this method for the identification of anti-SPI-2 inhibitors specific to <i>Salmonella</i> Typhimurium, which may be modified to investigate other virulence factors. For complete details on the use and execution of this protocol, please refer to Tsai et al. (2020).STAR protocolsHigh-Throughput Chemical Screening for Inhibitors of <i>Salmonella</i> Pathogenicity Island 2.PMC7580248FRN-156361FRN-15636Tsai CNCoombes BKfalseHigh-Throughput Chemical Screening for Inhibitors of <i>Salmonella</i> Pathogenicity Island 2.Anti-virulence therapies are under active investigation as antibiotic alternatives; however, their identification from large-scale chemical libraries poses a unique challenge. The dispensability of virulence factors for growth <i>in vitro</i> precludes conventional, optical density-based screening methods. Here, we provide a protocol for high-throughput screening with a cell-based, promoter reporter platform. We describe the use of this method for the identification of anti-SPI-2 inhibitors specific to <i>Salmonella</i> Typhimurium, which may be modified to investigate other virulence factors. For complete details on the use and execution of this protocol, please refer to Tsai et al. (2020).2020-01-01T00:00:00Z2020 Sep2025-05-29T19:38:10.003Z2025-05-29T19:38:10.003ZS-EPMC75802483311110010.1016/j.xpro.2020.100057