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Spectral Domain Optical Coherence Tomography (SD-OCT) applied to the mouse retina has been limited due to inherent movement artifacts and lack of resolution. Recently, SD-OCT scans from a commercially available imaging system have yielded retinal thickness values comparable to histology. However, these measurements are based on single point analysis of images. Here we report that using the Spectralis HRA + OCT Spectral Domain OCT and Fluorescein Angiography system (Heidelberg Engineering, Heidelberg, Germany), retinal thickness of linear expanses from SD-OCT data can be accurately assessed. This is possible by the development of a Spectralis-compatible ImageJ plug-in that imports 8-bit SLO and 32-bit OCT B-scan images, retaining scale and segmentation data and enabling analysis and 3D reconstruction. Moreover, mouse retinal layer thickness values obtained with this plug-in exhibit a high correlation to thickness measurements from histology of the same retinas. Thus, use of this ImageJ plug-in results in reliable quantification of long retinal expanses from in vivo SD-OCT images.