Characterization of the topmost surface of biomaterials is crucial to understanding their properties and interactions with the local environment. In this study, the oxide layer microstructure of plasma-modified 316L stainless steel (SS316L) samples was analyzed by a combination of electron backscatter diffraction and electron channeling contrast imaging using low-energy incident electrons. Both techniques allowed clear identification of a nano-thick amorphous oxide layer, on top of the polycrystalline substrate, for the plasma-modified samples. A methodology was developed using Monte Carlo simulations combined with the experimental results to estimate thickness of the amorphous layer for different surface conditions. X-ray photoelectron spectroscopy depth profiles were used to validate these estimations.