To investigate the diagnostic value of pepsin test in detecting laryngopharyngeal reflux (LPR) in patients with suspected LPR-induced dysphonia. Dysphonic and non-dysphonic patients with LPR at the 24-hour hypopharyngeal-esophageal impedance-pH monitoring (HEMII-pH) were recruited from January 2019 to November 2022. Patients collected saliva/sputum samples to measure pepsin concentrations. Symptoms and findings were studied through reflux symptom score (RSS) and reflux sign assessment (RSA). Voice quality was assessed with maximum phonation time, GRBAS, voice handicap index (VHI), and acoustic parameters at baseline and 3-month post-treatment. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of pepsin tests for dysphonia-related to LPR were calculated at ≥16ng/mL cutoff. The relationship between HEMII-pH, clinical features, voice quality outcomes, and pepsin measurement was investigated. Sixty-seven patients with LPR at the HEMII-pH completed the evaluations accounting for 30 patients consulting for dysphonia. Dysphonic patients reported higher RSS than non-dysphonic patients. RSS, RSA, laryngeal findings, VHI, and grade of dysphonia significantly improved from baseline to 3-month posttreatment. Pepsin test detected LPR in 73% of dysphonic cases. The pepsin test was 73.3 sensitive and 18.9 specific when considering the highest pepsin level of morning, postlunch, and postdinner sputum collections. Sensitivity, specificity, PPV, and NPV varied regarding the time of sputum collections. There was a strong significant association between the concentration of the morning pepsin test and the severity of laryngeal RSA score (P=0.018). The morning pepsin saliva test concentration was predictive of the 3-month otolaryngological RSS (P=0.014). Pepsin test is a sensitive but poorly specific diagnostic approach for patients with dysphonia attributed to LPR. Multiple pepsin measurements may increase the sensitivity and predictive value of pepsin test. Future large-cohort studies are needed to investigate the accuracy of pepsin test in this population.