In this study, we report the ability of anterior segment optical coherence tomography (AS OCT) in imaging the sub-Tenon space and its clinical application. High-speed AS OCT (Carl Zeiss Meditec, Dublin, Calif., USA) was used to visualize the sub-Tenon space during sub-Tenon injection. The sub-Tenon spaces, Tenon thickness, conjunctiva-Tenon thickness, injecting cannula position in relation to the sub-Tenon space and drug localization/distribution in the sub-Tenon space were analyzed. The sub-Tenon spaces of 12 of 11 patients were visualized with OCT during sub-Tenon injection up to 10-13 mm from the limbus. The mean conjunctiva-Tenon and Tenon thickness were 0.38 ± 0.08 and 0.21 ± 0.07 mm, respectively. The drug was tracked as bright white fluid. There was no conjunctival chemosis, subconjunctival drug or scleral perforation. Anterior segment OCT can be used for imaging the sub-Tenon space, especially during depot injections for confirmation of drug localization.