Pseudomonas aeruginosa chronically infects the lungs of people with cystic fibrosis (pwCF), where it produces a broad range of proteases that impact host immunity and inflammation. While some studies suggest these enzymes induce inflammation and may contribute to the excessive inflammatory response in pwCF, others indicate that they degrade cytokines and suppress the immune response. Here, we investigate the role of P. aeruginosa proteases in chronic CF lung inflammation by culturing clinical isolates under physiologically relevant conditions (synthetic CF sputum medium) and studying the inflammatory response in an organotypic 3-D lung cell culture model. Exposure to supernatants from isolates with proteolytic and elastolytic activity led to cytokine degradation and reduced cytokine release in the 3-D lung model. To identify the specific protease(s) responsible for the observed degradation effects, we performed a proteomics analysis on the supernatants of the P. aeruginosa CF isolates with and without proteolytic and elastolytic activity. Using functional annotation, 79 proteases were identified in the supernatants of the clinical isolates, with extracellular proteases among the most differentially expressed in the proteolytically active isolate group. The metalloprotease Elastase B (LasB) exhibited the highest level of differential expression. Therefore, we determined the specific contribution of LasB to the observed cytokine degradation by utilizing P. aeruginosa lasB knockout mutants. Compared to the lasB mutant, the wild-type strain significantly decreased inflammation by degrading key lung cytokines including MCP-1, IL-1β, GM-CSF and IL-8. While this study brings to light a key role for LasB, our findings demonstrate that immunomodulation by P. aeruginosa most likely involves the combined action of multiple proteases. This study provides a foundation for future research into protease-mediated immune evasion during chronic infection.