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Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.