Bacteria such as Staphylococcus aureus are generally engulfed by the host with a membrane when internalized by non-professional phagocytic host cells. These vacuoles then mature to phagosomes which can fuse with lysosomes enabling killing and digestion of the pathogen. Former proteome approaches enriching S. aureus from lysed host cells after infection did not cover secreted virulence factors. However, secreted S. aureus proteins should be trapped within the phagosomes and, hence, these compartments were enriched from human bronchial epithelial S9 cells infected with S. aureus HG001 to also explore the virulence factor repertoire of S. aureus following internalization by host cells. Using shotgun mass spectrometry we monitored 92 phagosomal proteins over time unraveling a heterogeneous composition of phagosomal proteins in regard to maturation stages. Furthermore, we quantified 36 bacterial proteins within the phagosomes during the first 6.5 h post infection by applying targeted single reaction monitoring. Among them were secreted bacterial virulence factors like lipases, hemolysins, and secreted adhesins which are usually hard to detect from infected sample material by proteomics approaches due to low abundance. Thus, we provide insight into the fate and early adaptation of S. aureus HG001 after internalization by epithelial cells by simultaneous analysis of the phagosomal environment and virulence factors of S. aureus.