Optical coherence tomography, an imaging modality using near-infrared light, produces cross-sectional tissue images with a lateral pixel resolution of 10 microm. However, normative data is first needed on epithelial thickness for lesion characterisation, and, to date, little exists. The purpose of our study is to measure normal laryngeal epithelial thickness by in vivo optical coherence tomography, and compare these values to those obtained from fixed ex-vivo laryngectomy specimens.Prospective at a single medical center in California, United States.A total of 116 patients undergoing operative endoscopy.Optical coherence tomography images of clinically normal laryngeal subsites were selected. Calibrated measurements of epithelial thickness at various laryngeal subsites were recorded. Measurements of epithelial thickness from corresponding areas were obtained using optical micrometry on histologically normal regions of 15 total laryngectomy specimens. Descriptive statistics were performed.Mean epithelial optical coherence tomography thicknesses were: true vocal cords (81 microm), false vocal cords (78 microm), subglottis (61 microm), aryepiglottic folds (111 microm), laryngeal epiglottis (116 microm) and lingual epiglottis (170 microm). Epithelial thicknesses in fixed tissues were: true vocal cords (103 microm), false vocal cords (79 microm), aryepiglottic folds (205 microm) subglottis (61 microm), laryngeal epiglottis (38 microm) and lingual epiglottis (130 microm).Optical coherence tomography does not have the artifacts associated with conventional histologic techniques. The inevitable development of office-based optical coherence tomography devices will increase the precision of laryngeal measurements and contribute to the clinical application of this technology in diagnosing laryngeal disease.