PURPOSE: To determine the efficacy of interferometry for examining the spatial changes to the corneal biomechanical response to simulated intraocular pressure (IOP) fluctuations that occur after corneal cross-linking (CXL) applied in different topographic locations. METHODS: Displacement speckle pattern interferometry (DSPI) was used to measure the total anterior surface displacement of human and porcine corneas in response to pressure variations up to 1 mm Hg from a baseline pressure of 16.5 mm Hg, both before and after CXL treatment, which was applied in isolated topographic locations (10-minute riboflavin soak [VibeX-Xtra; Avedro, Inc], 8-minute ultraviolet-A exposure at 15 mW/cm2). Alterations to biomechanics were evaluated by directly comparing the responses before and after treatment for each cornea. RESULTS: Before CXL, the corneal response to loading indicated spatial variability in mechanical properties. CXL treatments had a variable effect on the corneal response to loading dependent on the location of treatment, with reductions in regional displacement of up to 80% in response to a given pressure increase. CONCLUSIONS: Selectively cross-linking in different topographic locations introduces position-specific changes to mechanical properties that could potentially be used to alter the refractive power of the cornea. Changes to the biomechanics of the cornea after CXL are complex and appear to vary significantly depending on treatment location and initial biomechanics. Hence, further investigations are required on a larger number of corneas to allow the development of customized treatment protocols. In this study, laser interferometry was demonstrated to be an effective and valuable tool to achieve this.