A substrate inspection system includes a substrate support, optics configured to irradiate a patterned structure on the substrate and capture images of the patterned structure from light reflected from the patterned structure, a focus adjustment operative to adjust a focal position of the incident light on the patterned structure, and an image processor configured to calculate an optimal value of a focus offset used to establish focal points of the light for defect detection in the patterned structure. The patterned structure may include a first pattern having an opening and a second pattern having top surfaces located at different heights relative to the substrate. The value of the focus offset is determined using images of the top surfaces of the second pattern obtained while changing the focal position of the incident light.