Background: The pepsin test is an emerging non-invasive diagnostic approach for laryngopharyngeal reflux (LPR). The aim of this study was to investigate the diagnostic value of multiple salivary pepsin tests for detecting LPR. Methods: Patients with suspected LPR and asymptomatic individuals were consecutively recruited from January 2020 to November 2022. Patients benefited from hypopharyngeal–esophageal impedance-pH monitoring (HEMII-pH) and fasting and bedtime saliva collections to measure oral pepsin. The sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were calculated considering fasting, bedtime, and the highest values of the pepsin tests at ≥16, ≥36, ≥45, and ≥100 ng/mL cutoffs. Results: The pepsin test was adequately performed in 147 LPR patients and 32 controls. The pepsin tests were 81.6%, 74.8%, and 61.5% sensitive at cutoffs of ≥16, ≥45, and ≥100 ng/mL, respectively. The PPVs were 93.0%, 94.0%, and 94.8%, respectively. The highest specificity (81.8%) was found for the fasting pepsin test at a cutoff of 100 ng/mL. The highest sensitivity (81.6%) was found by considering the highest measured pepsin test at the ≥16 ng/mL threshold. The measurement of fasting saliva pepsin was associated with the highest sensitivity and specificity value. At ≥16 ng/mL, 27 patients had negative findings, indicating that 18.4% (27/147) of the true positive cases were missed by considering the highest pepsin test. The receiver operating characteristic curve reported that a cutoff of 21.5 was 76.9% sensitive and 62.5% specific, while the PPV and NPV were 91.1% and 38.2%, respectively. Conclusions: The consideration of the highest concentration of the fasting and bedtime saliva pepsin collections at a cutoff of 21.5 was associated with the best detection rate and sensitivity of the pepsin tests.