Staphylococcus aureus infection can disseminate systemically, causing inflammation, tissue damage, and metabolic alterations in multiple organs. This study aims to characterize region-specific metabolic changes in the liver, heart, spleen, and kidneys of S. aureus-infected mice using GC-Orbitrap and timsTOF analytical platforms. In preliminary experiments optimizing sample parameters across organs with distinct metabolic profiles, 20 tissue sections from the heart and kidneys yielded the highest number of detected features and the largest total peak area compared to 5 or 10 sections. However, this increase was not observed in other organs, indicating that organ-specific characteristics and sample preparation affect analytical sensitivity. Finally, we compared the metabolomic profiles of infected and uninfected mice to identify infection-associated metabolic pathways and candidate biomarkers, establishing a basis for understanding the metabolic signatures of S. aureus infection during systemic organ failure.