To determine the feasibility of using a handheld spectral-domain optical coherence tomography (SD-OCT) instrument to characterize normal corneal, retinal, and optic nerve head anatomy in vivo in standing horses. Clinically normal horses under sedation, palpebral nerve blockage, and pharmacologically induced mydriasis were imaged with a SD-OCT instrument (Envisu SD-OCT, Bioptigen, Inc., Morrisville, NC). Radial volumes from the cornea (axial, superior, inferior, nasal, and temporal), and rectangular volumes from the retina (dorsal, ventral, nasal, and temporal) and optic nerve head were acquired. Manual measurements of the corneal layers within the five regions, retinal and nerve fiber layer thickness in the four different regions adjacent to the ONH, and vertical and horizontal axis of the optic nerve head (ONH) and optic cup (OC) were obtained using the same device. Total corneal thickness (mean ± SD) measurements were 800 ± 50, 937 ± 61, 956 ± 61, 912 ± 65, and 884 ± 68 μm for the axial, superior, inferior, nasal, and temporal regions, respectively. The highest total retinal and nerve fiber layer thickness (mean ± SD), at the level of the ONH, was found nasally 459 ± 115 and 377 ± 116 μm, respectively, followed by the temporal, dorsal, and ventral quadrants. The dimensions of the ONH and OC (mean ± SD) were 3.682 ± 0.276 and 2.175 ± 0.502 mm for the horizontal, and 3.012 ± 0.278 and 2.035 ± 0.488 mm for the vertical axis. The SD-OCT instrument employed in this study may be used on sedated horses and allows the acquisition of high-resolution images, and thickness measurements involving the cornea, retina, and optic nerve.