We demonstrate that reflectance difference spectroscopy (RDS) is sensitive to defects induced by ion bombardment, located either in the topmost layer or in the subsurface region. Most importantly, these two kinds of defects can be spectrally discriminated, since the corresponding signatures in the RD spectrum arise from perturbations of different types of electronic states: The defects in the topmost surface layer mainly lead to a quenching of the optical anisotropy related to surface states, whereas the subsurface defects strongly affect the optical anisotropy originating from transitions between surface-modified bulk electronic states. Consequently, RDS can be used to simultaneously monitor the defects in the topmost surface layer and in the subsurface region in-situ during ion bombardment and thermal annealing.Characteristic RD spectra and the corresponding STM images for a Cu(110) substrate before and after healing of the subsurface defects.