The aim was to investigate intracorneal iontophoresis of biolabile triamcinolone acetonide (TA) amino acid ester prodrugs (TA-AA). Arginine and lysine esters of TA (TA-Arg and TA-Lys, respectively) were synthesized and characterized; quantification was performed by HPLC-UV and UHPLC–MS/MS. The aqueous solubility of the prodrugs (at pH 5.5) was ˜1000-fold greater than TA. Anodal iontophoresis (10 min at 3 mA/cm2) of TA-AA was investigated using isolated porcine cornea. Although no statistically significant difference was observed in total intracorneal delivery of TA (468.25 ± 59.70 and 540.85 ± 79.16 nmolTA/cm2, for TA-Arg and TA-Lys, respectively), the different susceptibilities of the prodrugs to hydrolysis influenced intracorneal biodistribution. Quantification of TA in twenty-five 40 μm thick corneal lamellae revealed significantly deeper penetration of TA following TA-Lys iontophoresis. Its superior resistance to hydrolysis enabled sustained electromigration into the deeper cornea suggesting judicious prodrug selection might enable targeted regioselective drug delivery. The intracorneal biodistribution following anodal iontophoresis of TA-Arg (2.3 mM; 10 min, 3 mA/cm2) was visualized by full field optical coherence tomography providing qualitative confirmation of the extensive intracorneal penetration of TA. Short duration iontophoresis of TA-AA prodrugs may improve deep corneal bioavailability and efficacy in vivo, constituting a “single-shot” treatment option for corneal allograft rejection.