Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses and causing host cell death. Classical methods assessing S. aureus cytotoxicity are limited and do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (Intracellular Toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Using InToxSa, a versatile and high-throughput cell-based phenomics platform, we have identified clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.