Focal distance directly impacts endothelial cell counting accuracy in specular microscopy. Images were taken of a calibration lens at focal distances from 0-100μm from baseline. Greater focal deviation led to larger measured cell areas and lower calculated cell densities, with errors becoming non-negligible above 30μm and problematic above 60μm deviation. Higher resolution imaging allows better assessment of focal distance effects. Maintaining precise focus is essential for accurate cell density measurements.