PURPOSE. To assess the accuracy of human blood oximetry measurements in a model eye with a hyperspectral fundus camera. METHODS. Seven human whole blood samples (two arterial, five venous) were obtained, the oxygen saturations measured with a CO oximeter, and the samples inserted into quartz tubes with internal diameters of 100 and 150 µm. The tubes (n = 20; ten 100 µm and ten 150 µm) were placed within a model eye in front of a background reflectance surface with reflectivities of 20%, 60%, and 99%. Spectral images at wavelengths between 500 and 650 nm were acquired with a hyperspectral fundus camera and analyzed with an oximetric model to calculate the oxygen saturation of blood within the tubes. The calculated oxygen saturations were compared with the measured oxygen saturations. The effects of the background reflectivity and tube size on the accuracy of the calculated oxygen saturations were evaluated. RESULTS. Background reflectivity and tube size had no significant effect on the mean oxygen saturation difference (P = 0.18 and P = 0.99, respectively; repeated-measures, two-way ANOVA). The mean differences (SD) between the measured and calculated oxygen saturations in segments of the 100 and 150 µm tubes overlying the 20%, 60%, and 99% background reflectivities were (100 µm) -4.0% (13.4%), -6.4% (9.9%), and -5.5% (10.2%) and (150 µm) -5.3% (10.8%), -5.2% (10.7%), and -5.2% (10.9%), respectively. CONCLUSIONS. There was reasonable agreement between the measured oxygen saturation values and those calculated by the oximetry model. The oximetry model could be used to determine the functional health of the retina. © 2011 The Association for Research in Vision and Ophthalmology, Inc.