This protocol provides details on the cell dissociation that should be performed to obtain single-cell suspensions from airway biopsies.Biopsies may come from tracheal, bronchial or nasal epithelium.Cell dissociation is performed at 4°C to avoid gene expression alterations and maximize viability. The typical cell number recovery is 40 000 cells for one biopsy.Cell suspensions are suitable for single-cell RNA-sequencing protocols.