Background. There is no gold standard test for diagnosis of gastroesophageal reflux disease (GERD) associated infantile wheezing. Objectives. To evaluate the value of bronchoalveolar lavage (BAL) pepsin assay in diagnosis of GERD in wheezy infants. Methods. Fifty-two wheezy infants were evaluated for GERD using esophageal combined impedance-pH (MII-pH) monitoring, esophagogastroduodenoscopy with esophageal biopsies, and BAL pepsin. Tracheobronchial aspirates from 10 healthy infants planned for surgery without history of respiratory problems were examined for pepsin. Results. Wheezy infants with silent reflux and wheezy infants with typical GERD symptoms but normal MII-pH had significantly higher BAL pepsin compared to healthy control (45.3 ± 8.6 and 42.8 ± 8 versus 29 ± 2.6, P < 0.0001 and P = 0.011, resp.). BAL pepsin had sensitivity (61.7%, 72 %, and 70%) and specificity (55.5%, 52.9%, and 53%) to diagnose GERD associated infantile wheeze compared to abnormal MII-pH, reflux esophagitis, and lipid laden macrophage index, respectively. Conclusion. A stepwise approach for assessment of GERD in wheezy infants is advised. In those with silent reflux, a trial of antireflux therapy is warranted with no need for further pepsin assay. But when combined MII-pH is negative despite the presence of typical GERD symptoms, pepsin assay will be needed to rule out GERD related aspiration.