This study employed tandem mass tag (TMT) quantitative proteomics to investigate the dose-dependent effects of Staphylococcus aureus virulence factor phenol-soluble modulin α3 (PSMα3) on the global protein expression profile of human macrophages. THP-1 monocytic cells were differentiated into macrophages using PMA and then treated with 0 (control), 1, or 3 μM PSMα3 for 24 hours. Cellular proteins were extracted, digested, labeled with TMT reagents, and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on an Orbitrap Exploris 480 platform. The dataset aims to identify proteins and pathways significantly altered by PSMα3 exposure, providing insights into the molecular mechanisms underlying macrophage responses to staphylococcal infection and potential targets for therapeutic intervention.