Staphylococcus aureus is a major pathogen, causing significant morbidity, mortality, and healthcare-associated costs, complicated further by increasing antimicrobial resistance. Macrophages, the resident tissue phagocyte are essential for bacterial clearance. S. aureus is rapidly internalised by macrophages, but phagosomal maturation is subverted to establish an intracellular population. Phagosomal maturation after bacterial ingestion normally involves sequential fusion with endosomes and lysosomes with reducing luminal pH, facilitating degradation of bacteria. In response to phagocytosis and phagosomal maturation, S. aureus gene expression adapts to the intracellular environment with up-regulation of multiple genes involved in resistance to oxidative stress and global regulation. A greater understanding of intracellular persistence is required to develop more effective treatment of S. aureus. The first aim of this project was to confirm that S. aureus USA300 strain JE2 demonstrates this phenotype. Using an in vitro differentiated macrophage model, phagosomes containing the USA300 strain JE2 progress to a late phagosome state but fail mature to a phagolysosome state and to acidify appropriately. The second aim was to develop a high-throughput microscopy protocol to screen intracellular bacterial acidification in macrophages. This enabled the final aim of assessing a USA300 strain JE2-derived library of transposon-mutated non-essential genes to identify genes associated with phagosomal acidification subversion. This novel screening tool can also be repurposed to investigate other pathogens to broaden knowledge of host:pathogen interaction. A total of 15 genes were identified, including the regulators agr and saeR, and oxidative stress enzyme catalase. The protease ClpP, the terminal oxidase complex genes qoxA and qoxC, and the cytochrome assembly complexes ctaB and ctaM were novel findings in context of phagosomal acidification subversion. A greater understanding of these genes and their regulatory pathways will offer insight into the mechanisms utilised by S. aureus to subvert the host macrophage immune response and aid novel therapeutic target development.