Background: Neutrophilic airway inflammation is frequently observed in severe uncontrolled asthma as well as in controlled asthma. However, there is no sputum biomarker to differentiate the two conditions. Objective: To identify biomarkers of severe uncontrolled asthma with neutrophilic airway inflammation. Methods: Sputum with a ＞70% neutrophil content was pooled from 5 severe uncontrolled asthmatics (UA) and from 10 controlled asthmatics (CA). 2-DE was adopted for differential display proteomics, and candidate proteins were identified using MALDI-TOF MS analysis. S100 calcium binding protein A9 (S100A9) was identified by western blot and its level was measured in sputum from asthmatics with varying disease severity, COPD, and normal controls, by ELISA. Results: 14 protein spots exhibited differences in intensity between severe UA and CA. MALDI-TOF/TOF of these spots revealed an increase in HNP- 2, S100A9, α-amylase, NGAL, 4-aminobutyrate transaminase, and cystatin SA in patients with UA compared to patients with CA. There was a decrease in PLUNC, C3 component, immunoglobulin heavy chain variable region, GFAP1, IgM kappa IIIb SON, MLL-AF4 der (11) fusion protein, cytokeratin 8, and recombinant IgG4 heavy chain. S100A9 was detected at a higher level in western blots of neutrophilic sputum from UA, and significantly increased as measured by ELISA, in neutrophilic UA compared to CA, and COPD. Conclusions: S100A9 in sputum may be a biomarker of neutrophilic inflammation in severe uncontrolled asthma.