Laryngopharyngeal reflux (LPR) is often considered to be a contributory factor to the development of a spectrum of laryngeal abnormalities including vocal cord leukoplakia and dysplasia. This is especially the case when traditional risk factors, such as tobacco smoking, are absent. Proving that LPR is responsible is, however, more of a challenge. Pepsin has recently been reported to be a biomarker of LPR, and the aim of this paper was to assess for the presence of pepsin in vocal fold biopsies using immunohistochemistry (IHC). Twenty-six patients with laryngeal leukoplakia that was histologically identified to represent epithelial hyperplasia were enrolled. The results of IHC for pepsin in their biopsy samples were compared to a control group of 20 normal vocal folds collected from autopsies within six hours post-mortem. IHC for pepsin was positive in 17/26 (65%) of the patients with vocal fold leukoplakia, compared to 4/20 (20%) of the control patients (p < 0.001). Of the 17 subject patients with pepsin positivity, 11 were weakly positive, four were positive and two were strongly positive. The four control patients with IHC positivity were all weakly positive only. Although this is only a retrospective pilot study with a limited number of patients, the results are certainly promising and justify further research into the utility of IHC for pepsin in laryngeal biopsy samples to guide adjuvant medical treatment for patients with laryngeal leukoplakia. Having an objective test for LPR would be especially helpful when counselling patients with laryngeal abnormalities who are not infrequently skeptical about reflux being the cause.