To study the presence of relative afferent pupillary defect (RAPD) in patients with glaucoma with the help of a custom-built pupillometer.Sixty-five participants were recruited (32 with open-angle glaucoma and 33 healthy subjects). All underwent standard clinical examination including perimetry and optic disc photography. Pupillary light reflexes were examined with a custom-built pupillometer. Three video sequences were recorded for each subject. Alternating light stimulation with a duration of 0.5 seconds was used, followed by a 1 second pause. Mean values of pupil area ratio (PAR), pupil contraction velocity ratio (PCVR), and pupil dilation velocity ratio (PDVR) were calculated. Receiver operating characteristic (ROC) curves were constructed for each of the three parameters. Intra-individual variability was estimated.PAR and PDVR differed significantly between the group with glaucoma and the control group (P < 0.0001). PAR was more sensitive for glaucoma detection than the other pupillometric parameters (PCVR and PDVR). The area under the receiver operating characteristic curve was largest for PAR. At a fixed specificity of 90%, sensitivity for PAR was 86.7%.Measuring RAPD with infrared computerized pupillometry can detect optic neuropathy in glaucoma with high sensitivity and specificity. The method is fast and objective. Pupil area amplitude measurements were superior to pupil velocity measurements for the detection of RAPD in glaucoma.