To investigate near infrared iris transillumination (NIRit) imaging as a new method to quantify pupil shape, size, and position because the imaging modality can uniquely provide simultaneous information regarding iris structural details that influence pupil characteristics and because exploration of related techniques could promote discovery helpful to clinical research and care. Digital NIRit images of normal and diseased eyes were used along with computer-assisted techniques to quantify four primary pupil parameters, including pupil roundness (PR), pupil ovalness (PO), pupil size (PS), and pupil eccentricity (PE). A combined measure of PR and PO was also developed (the pupil circularity index [PCI]). Repeatability of the measures was studied and example analyses were performed. Pupil measures could be calculated for right eyes of 307 subjects (164 normal, 143 other), with fewer than 0.5% exclusions due to image quality. Repeatability study did not show significant bias (P < .05) for any of the four primary measures. Example analyses could show age-associated differences in pupil shape (≥ 50 year olds had less regular pupils than < 50 year olds: median PCI = 0.009 vs 0.006; P < .01) and that a group of pigment dispersion syndrome subjects (n = 27) had less regular pupils than a group of matched controls (PO = 0.9966 vs 0.9990; P < .05). Digital NIRit imaging can provide novel, reliable, and informative methods to quantify pupil characteristics while providing simultaneous information about iris structure that may influence these parameters.