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It is well established that the state of corneal hydration plays a crucial role in maintaining optimal vision. Therefore, any knowledge that can be obtained non-invasively about the status of corneal hydration could be of significant clinical value. A novel confocal Raman spectroscopic technique was used to monitor non-invasively drug-induced hydration changes in the rabbit cornea. The spectroscopic technique enables one to monitor the changes in water content of the cornea while the confocal probing reduces interference of signals from adjacent tissues and allows for measurement of corneal hydration at various depths. The corneal hydration is altered by applying a dehydrating agent (Muro 128^®) topically on the cornea. To determine the corneal hydration status, the OH/CH ratio between the Raman intensity of the water OH mode at 3390 cm⁻¹ and the protein CH stretching mode at 2945 cm⁻¹ is calculated. In the middle of the corneal stroma after 10 min, Muro 128^®-treated corneas show an average decrease of about 30% in the OH/CH ratio (1.27 ± 0.13) compared with the untreated corneas (1.76 ± 0.09). In this in vivo model it is possible to monitor the hydration status of the living cornea using the Raman spectroscopic technique. Copyright © 2001 John Wiley & Sons, Ltd.