Introduction. Although airway mucus dehydration is key to pathophysiology of cystic fibrosis (CF) and other airways diseases, measuring mucus hydration is challenging. We explored a robust method to estimate mucus hydration using sialic acid as a marker for mucin content. Methods. Terminal sialic acid residues from mucins were cleaved by acid hydrolysis from airway samples, and concentrations of sialic acid, urea, and other biomarkers analyzed using mass spectrometry. Results. In mucins purified from human airway epithelial (HAE), sialic acid concentrations after acid hydrolysis correlated with mucin concentrations (r2=0.92). Sialic acid/urea ratios measured from filters applied to the apical surface of cultured HAE correlated to % solids and were elevated in samples from CF HAEs relative to controls (2.2±1.1 vs. 0.93±1.8, p<0.01). Sialic acid/urea ratios were elevated in bronchoalveolar lavage fluid (BALF) from βENaC transgenic mice, known to have reduced mucus hydration, and mice sensitized to house dust mite allergen. In a translational application, elevated sialic acid/urea ratios were measured in BALF from young children with CF who had airway infection relative to those who did not (5.5±3.7 vs.1.9±1.4, p<0.02), and could be assessed simultaneously with established biomarkers of inflammation. Conclusions. The sialic acid/urea ratio performed similarly to % solids, the gold standard measure of mucus hydration. The method proved robust and has potential to serve as flexible techniques to assess mucin hydration, particularly in samples like BALF in which established methods such as % solids cannot be utilized. -