Purpose: To determine the thickness of Bowman's layer (BL) in vivo in a normal, healthy population, and to determine its variation with age.Methods: 82 subjects aged 15 -88y with clear, healthy corneas were examined bilaterally with laser-scanning in vivo confocal microscopy (IVCM). BL thickness was determined from IVCM images of anterior and posterior BL boundaries. For a given eye, BL thickness was averaged across four central locations by two independent observers. Additionally, central corneal thickness was measured by time-domain optical coherence tomography (OCT).Results: A significant negative correlation of BL thickness with age was found in both right (Pearson r = -0.579, P < 0.0001) and left (r = -0.558, P < 0.0001) eyes. Linear regression analysis yielded a decline in BL thickness of 0.06 µm/year. Older subjects (mean 64.4y; N = 41) had a mean BL thickness (8.6 ± 1.7 µm, RE) significantly thinner than younger subjects (mean 31.6y; N = 41; 10.7 ± 1.6 µm, RE; P < 0.001). No correlation of corneal thickness with age or BL thickness with corneal thickness was found. Strong inter-eye correlations in BL thickness (Pearson r = 0.771, P < 0.0001) and corneal thickness (r = 0.969, P < 0.001) were found.Conclusions: BL thins with age in the normal cornea, losing one-third of its thickness between the ages of 20 and 80. In vivo measurement of BL thickness by IVCM could aid in clinical assessment and planning treatments of the anterior cornea.3