Viewing the scarce amount of protein material coming from the bacterial pathogen in infection models and despite the availability of contemporary, highly sensitive and fast scanning mass spectrometers, the power requirement still not suffices to study the host and pathogen proteomes simultaneously. In the present work we aimed to establish a DIA mass spectrometry workflow for improving the protein identification and quantification of LC-MS/MS, particularly in case of complex samples containing a fairly low amount of peptide material derived from Salmonella, therefore enabling simultaneous host and pathogen protein expression profiling reflecting actual infection conditions.