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Summary: The release of neutrophil extracellular traps (NETs) has been involved in numerous infectious and non-infectious diseases. Nevertheless, quantitative analysis of NETs in vivo has been challenging. Here, we present a protocol for NET quantification by flow cytometry in the bronchoalveolar lavage fluid (BALF) of mice upon pulmonary infection with S. aureus. We describe steps for bacteria growth and instillation and BALF recovery. We then detail staining to quantify the release of NETs and neutrophils recruited to the site of infection.For complete information on the generation and use of this protocol, please refer to Poli et al. (2021)1 and Poli et al. (2022).2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.