So far the study of chemical burns has lacked techniques to define penetration kinetics and the effects of decontamination within biological structures. In this study, we aim to demonstrate that high-resolution optical coherence tomography (HR-OCT) can close this gap. Rabbit corneas were exposed ex vivo to 2.5% hydrofluoric acid (HF) solution, and microstructural changes were monitored in the time domain by OCT imaging. HF application and penetration resulted in shrinkage of the corneal thickness, interpreted as a result of osmolar changes and of loss of water-binding capacity, and a substantial increase in OCT signal amplitudes. The effectiveness of different rinsing solutions on the chemical burn was also evaluated. With tap water and with 1% calcium gluconate, the deep corneal stroma remained clear until the end of the rinsing period but became opaque afterwards. With Hexafluorine, the cornea remained clear for 60 min after rinsing ceased. We conclude that HR-OCT can assist in the clinical evaluation of an ex vivo eye irritation test, and that decontamination of an HF burn using Hexafluorine is efficient.