To apply software-based image-analysis tools to objectively determine intraocular scatter determined from clinically derived Hartmann-Shack patterns. Aston Academy of Life Sciences, Aston University, Birmingham, United Kingdom, and Department of Optics, University of Valencia, Valencia, Spain. Purpose-designed image-analysis software was used to quantify scatter from centroid patterns obtained using a clinical Hartmann-Shack analyzer (WASCA, Zeiss/Meditec). Three scatter values, as the maximum standard deviation within a lenslet for all lenslets in the pattern, were obtained in 6 model eyes and 10 human eyes. In the model-eye sample, patterns were obtained in 4 sessions: 2 without realigning between measurements, 1 with realignment, and 1 with an angular shift of 6 degrees from the instrument axis. Three measurements were made in the human eyes with the C-Quant straylight meter (Oculus) to obtain psychometric and objective measures of retinal straylight. Analysis of variance, intraclass correlation coefficients, coefficient of repeatability (CoR), and correlations were used to determine intrasession and intersession repeatability and the relationship between measures. No significant differences were found between the sessions in the model eye (P=.234). The mean CoR was less than 10% in all model- and human-eye sessions. After incomplete patterns were removed, good correlation was achieved between psychometric and objective scatter measurements despite the small sample size (n=6; r=-0.831; P=.040). The methodology was repeatable in model and human eyes, strong against realignment and misalignment, and sensitive. Clinical application would benefit from effective use of the sensor's dynamic range.