Background: The Lyon Consensus classification confirms or rules out gastroesophageal reflux disease (GERD). The refractory symptoms of patients with GERD taking proton pump inhibitors (PPIs) are challenging in clinical practice. Salivary pepsin concentration was proposed as a diagnostic biomarker for GERD. We aimed to determine the diagnostic value of salivary pepsin concentration for patients with conclusive GERD, based on the Lyon classification, and the correlation of salivary pepsin concentration with parameters of high-resolution manometry and 24-h multichannel intraluminal impedance-pH in patients with PPI-refractory symptoms. Material/Methods: Saliva samples obtained from 130 patients who were suspicious for GERD and had PPI-refractory symptoms were used for pepsin determination using the enzyme-linked immunosorbent assay. All patients underwent upper gastrointestinal endoscopy, high-resolution manometry, 24-h multichannel intraluminal impedance, and pH recording and were classified as conclusive GERD, inconclusive GERD, and evidence against GERD groups according to Lyon classification. Results: Salivary pepsin concentration was 8.2 ng/mL (3.8-17.8 ng/mL), 4.0 ng/mL (2.3-6.1 ng/mL), and 2.4 ng/ml (2.2-3.1 ng/mL) in conclusive GERD, inconclusive GERD, and evidence against GERD groups, respectively (P<0.001), and had a negative correlation with distal mean nocturnal baseline impedance and positive correlations with acid exposure time, total number of reflux events, and esophagogastric junction type. The area under the ROC curve of salivary pepsin for conclusive GERD was 0.76 (0.68-0.84), with a sensitivity of 76.36% and a specificity of 63.41% for conclusive GERD diagnosis at a cut-off value of 4.21 ng/ml. Conclusions: Salivary pepsin test had moderate diagnostic value for conclusive GERD by Lyon classification in patients with PPI-refractory symptoms.