Objectives/Hypothesis To determine salivary pepsin levels as a function of collection time. Study Design A prospective, case‐control study. Methods We selected 57 patients with clinical symptoms and signs of laryngopharyngeal reflux who underwent 24‐hour multichannel intraluminal impedance‐pH (24h MII‐pH) monitoring tests, and 12 control subjects without clinical symptoms and signs of laryngopharyngeal reflux. All subjects were instructed to collect saliva samples upon waking, 1 hour after each meal (3 times per day), and upon any occurrence of laryngopharyngeal reflux (LPR) symptoms. The pepsin levels in saliva were measured by using enzyme‐linked immunosorbent assay. The Reflux Symptom Index survey was also administered. Results A total of 50 patients in whom LPR was diagnosed based on results of 24h MII‐pH monitoring test were enrolled in the study. The average pepsin level upon waking was 17.2 ng/mL, which was significantly higher than that measured in samples collected at any other time (P < .005). Pepsin levels were higher in patients with LPR than those in controls. The Reflux Symptom Index scores were statistically different between these groups of subjects. Conclusions The levels of total pepsin in saliva collected upon waking were significantly higher in the group of patients presenting with LPR symptoms and who demonstrated at least one episode of proximal esophageal reflux during 24h MII‐pH monitoring. Furthermore, the average pepsin level upon waking was higher than that measured at any other time. Measuring pepsin levels in the saliva upon waking may be a useful method in the diagnosis of LPR. Level of Evidence 3b Laryngoscope, 126:2770–2773, 2016