Mass spectrometry-based proteomics by bottom-up approaches enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying of dual biological systems in a single experiment, supporting investigation of infection from the perspective of both a host and pathogen. At the ocular surface, P. aeruginosa are commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, we profile the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa and detect neutrophil marker proteins, indicating neutrophil recruitment to the site