To identify the newly synthesized proteins by P. aeruginosa after engulfment by host immune cells, we employed the pulsed-SILAC proteomics approach to label the bacterial proteome with L-lysine-13C6 15N2 (Lys8) and L-lysine (Lys0) before and after entering host cells, respectively. The PAO1△lysA mutant which failed to synthesize lysine was cultured in ABTG medium 37 supplemented with stable isotope-containing amino acid L-lysine-13C6 15N2 (Lys8). The Lys8 labeled bacteria were then harvested and used to infect the RAW264.7 cells which were cultured in DMEM containing regular L-lysine (Lys0). After 4 h infection, RAW264.7 cells were lysed, and the intracellular bacteria were harvested and analyzed by LC-MS/MS. For the control DMEM group, labeled bacteria were diluted in pure DMEM medium. By using this pulsed-labelling approach, the newly synthesized proteins can be selectively tagged with Lys0 during the infection.