Spray application of Mycoplasma gallisepticum (MG) vaccines is a labor- and time-saving means of mass vaccination of layer chickens. Recent assessment of spray characteristics of nozzles commonly used to apply MG vaccine in layer chicken operations has shown that the amount of respirable droplets (<5 µm) is negligible. Topical application of vaccine onto the eye surface has been suggested as a route of vaccination, but no estimates of vaccine load delivered via spray application were found in the literature. Estimates of eye surface area were developed using digital imaging; 24 layer pullets were used for analysis, and the mean eye surface area, corrected for corneal curvature, was found to be 0.609 cm². This surface area was then used to estimate vaccine load for commercially available live MG vaccine sprayed through popular nozzles. Less than 3000 colony-forming units can be expected for direct deposition onto the surface of an eye.