Staphylococcus aureusinfections are associated with high mortality rates. Often considered an extracellular pathogen,S. aureuscan persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessingS. aureuscytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model,we have developed a platform calledInToxSa(intracellulartoxicity ofS. aureus) to quantify intracellular cytotoxicS. aureusphenotypes. Studying a panel of 387S.aureusbacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations inS. aureusclinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations inausA, encoding the aureusimine non-ribosomal peptide synthetase, reducedS. aureuscytotoxicity, and increased intracellular persistence.InToxSais a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevantS. aureuspathoadaptive mutations that promote intracellular residency.