In this study, we used an interdisciplinary approach, combining biophysics and systems biology, to design a microfluidic chip for the multiplexed detection and separation of pathogen cells from immune cells combined with proteomic profiling using mass spectrometry. Our nanochip design includes precise control of the microfabricated filters pore size for exclusion of cell types based on size to reliably separate bacteria from immune cells (REF 20, 21 NFRF), enabling separation of the pathogen into distinct populations. Our results show consistency in macrophage cell proteomes using a traditional ‘cell scraping’ vs. nanochip collection of host cells, demonstrating minimal disruption to the infectome profile. However, comparison between ‘cell scraping proteome profiling and nanochip proteome profiling reveals deeper coverage of the bacterial proteome, a key component when investigating regulators of bacterial virulence. Additionally, separation of K. pneumoniae cells into non-phagocytosed vs. phagocytosed (or associated) distinct populations provides new biological insights into mechanisms of immune cell evasion used by the bacterium to promote survival within the host . This information presents a plethora of new targets for inhibition or perturbation to weaken the bacterium and enhance clearance by the host. Here, we present a reproducible and adaptable technological platform that combines the precision of microfluidics with the power of mass spectrometry-based proteomics to distinguish biological systems and to delve deeper into mechanisms regulating the outcome of infectious disease.