Updated project metadata. Staphylococcus aureus is a major contributor of healthcare-associated infections and able to withstand numerous frequently used antibiotics. In order to develop novel treatment approaches, a deeper understanding of the complex interaction between host and pathogen is required. This is particularly true for the processes during development of tissue abscesses, which are important contributors to acute and persistent staphylococcal infections. Notably, the formation of staphylococcal abscesses takes place in distinct stages. Consequently, studies aiming to elucidate the underlying mechanisms of abscess development have to take temporal and spatial components into account. State-of-the-art proteomic technologies offer insight into complex biological systems. However, the pairing of spatial information and deep proteomic analysis is challenging, complicating the targeted analysis of relatively small-scale and heterogeneous structures like organ abscesses. To overcome existing technical limitations, we have previously introduced the use of a spatially targeted liquid extraction surface analysis (LESA) workflow termed “microLESA”. In our current study, we perform microLESA to investigate the processes at the host-pathogen interface during development of staphylococcal kidney abscesses in a murine model of systemic infection. For this investigation, regions from the abscess community, the interface surrounding the abscess, and the cortex of infected kidneys were extracted and analyzed at both 4- and 10-days post infection. By defining the proteome of different abscess regions across the course of infection, we followed the immune response and bacterial contribution to abscess development through spatial and temporal proteomic changes. The information gathered was then mapped through pathway analysis to characterize the metabolic processes at the host-pathogen interface during staphylococcal infection.