The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H 2 O 2 ) and hypothiocyanite (OSCN - ), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The in vitro studies of the oxidative responses are performed mainly by one-shot H 2 O 2 exposure that does not recapitulate the complex H 2 O 2 /LPO/SCN - system releasing the reactive oxygen species in airway secretions. A cell-free in vitro assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H 2 O 2 /OSCN - concentrations produced within this in vitro assay and assessed the resistance of Pseudomonas aeruginosa and Staphylococcus aureus clinical strains to the HAE oxidative response. We found that H 2 O 2 /OSCN - were steadily produced from 7h and up to 25h, but OSCN - was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H 2 O 2 of roughly 50% for 10 5 and 10 7 colony-forming unit (CFU)/mL inocula, while 10 2 and 10 4 CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to P . aeruginosa , S . aureus showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this in vitro assay can be helpful in assessing whether pathogens can resist the antibacterial oxidative HAE response. We anticipate these findings as a starting point for more sophisticated in vitro models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response.