Diagnosis of extraesophageal reflux (EER) currently relies on tools designed for diagnosis of gastroesophageal reflux. Such tools lack the sensitivity and reproducibility to detect the less frequent and mildly acidic reflux associated with upper airway disease. Pepsin has been posited to be a reliable biological marker of EER. Our aim was to present a comprehensive literature review of the use of pepsin as a diagnostic marker of EER. Two methods are typically used for detection of pepsin in the airways: enzymatic and immunologic. The limitations, advantages, and examples of use of each are discussed. Pepsin assay has been used to identify refluxate in trachea, lung, sinus, middle ear, combined sputum and saliva, and breath condensate. An immunologic pepsin assay of combined sputum and saliva was determined to be 100% sensitive and 89% specific for detection of EER (based on pH-metry), and an enzymatic test of nasal lavage fluid (100% sensitivity and 92.5% specificity) demonstrated an increased incidence of EER in patients with chronic rhinosinusitis. Pepsin assay identified tracheal pepsin to be an indicator of bronchopulmonary dysplasia and related mortality risk in ventilated preterm infants. Pepsin assay is a useful tool for correlation of reflux with airway disease and is a reliable diagnostic marker of EER.