The purpose of this study is to assess the possible application of second harmonic generation (SHG) microscopy for demonstrating the structural alterations of bovine cornea following riboflavin-UVA-induced cross-linking treatment, and to investigate its potential as being a clinical in vivo monitoring technique of cross-linking treatment. Riboflavin and UVA cross-linking treatment has been applied in the clinics for improving biomechanical strength in ecstatic corneal diseases including keratoconus and keratectasia following refractive surgery. Increasing the degree of cross-linking of stromal collagen may help to halt the progression of ecstatic changes of cornea. However, the efficacy and extension of current cross-linking protocol could not be quantified using current optical monitoring system. In this work, we attempted to use the non-invasive optical SHG imaging technique for demonstrating and quantifying the degree of cross-linking of stromal collagen in an experimental bovine corneal model. We found that significant curvy changes of parallel aligned corneal collagen fibers could be identified with SHG imaging. We proposed that SHG imaging can be an effective monitoring and quantifying system for evaluation of the efficacy of cross-linking treatment in the cornea.