Abstract: A method and system of acquisition and processing of data representing oxygen saturation (OS) value of a tissue of a visual system of a subject, such as the optic nerve head and overlying artery and vein. The data is acquired at pre-determined discrete spectral points, including at least two isosbestic points, as a discrete reflectance spectrum, with the use of a multi-spectral optical imaging system that simultaneously produces a plurality of two-dimensional spectrally-discrete images by segmenting an incoming light front with secondary objectives. The OS value is assessed based on determination of areas bound by an acquired discrete reflectance spectrum and an isosbestic line.