A substrate inspection method includes: (i) acquiring a plurality of defect of interest (DOI) images of a substrate having a DOI, under a corresponding plurality of different optical conditions, (ii) acquiring a plurality of DOI difference images from differences between the plurality of DOI images and a reference image, and (iii) acquiring a plurality of DOI difference-of-difference (DOD) images from differences between the plurality of DOI difference images. The method also includes setting two optical conditions corresponding to a DOI DOD image having the highest signal-to-noise ratio (SNR) among the plurality of DOI DOD images, as a first optical condition and a second optical condition, and acquiring a first image of the substrate under the first optical condition and a second image of the substrate under the second optical condition. A first difference image is also acquired, which is a difference between the first image and the reference image, and a second difference image is acquired, which is a difference between the second image and the reference image. A DOD image is acquired that is a difference between the first difference image and the second difference image. A low-SNR defect candidate region is then detected from the first difference image, the second difference image, and the DOD image.