Retinal ganglion cell (RGC) death induced by optic nerve crush injury has been used as a model to investigate axonal degeneration of the central nervous system in mice.1–4 In adult mice, approximately 60% of RGCs are lost within 3 weeks after optic nerve crush injury, as determined by histology.1,4 Ophthalmic imaging techniques such as confocal scanning laser ophthalmoscopy (CSLO) and optical coherence tomography (OCT) can provide quantitative and detailed structural information from the retina and optic nerve head (ONH) region. In vivo images of RGCs in mice and rats after optic nerve crush have been acquired using retrograde labeling and CSLO.5–7 Recently, Leung et al.8 demonstrated the use of CSLO with Thy-1 cyan fluorescent protein expressing transgenic mice to monitor RGCs in vivo after nerve crush. The authors in these studies were able to observe the loss of RGCs in the same eyes over time, contrary to previous experiments using histology that required several different animals at each time point. OCT allows for noninvasive, in vivo imaging of ocular structures and has been used to obtain optical cross sections of the mouse retina.9–12 Time-domain OCT9,10 and spectral-domain OCT (SD-OCT)13–17 imaging have been used to observe retinal thinning in mouse models of retinal degeneration. One study used SD-OCT to image axotomized rats and manually obtained measurements of retinal layers in six cross-sectional images.18 This approach, however, is prone to subjective bias both in the selection of individual cross-sections and in the manual segmentation of retinal layers. To fully benefit from the high resolution and rapid scanning provided by SD-OCT, it would be desirable to obtain automated measurements from the area of interest. The goal of this study, therefore, was to investigate the longitudinal effect of optic nerve crush injury on total retinal thickness (TRT) in adult mice using automated segmentation analysis of SD-OCT images.