Purpose To determine the sensitivity and specificity of various methods of detecting a relative afferent pupillary defect (RAPD) in patients with glaucoma-related diagnoses. Patients and methods Patients underwent RAPD evaluation using the swinging flashlight method (SFM), the magnifier-assisted SFM, and pupillography using the Konan RAPDx. Main outcome measures were sensitivity and specificity of three methods of RAPD evaluation in detecting visual field mean deviation (MD), cup to disc ratio (CDR), disc damage likelihood scale (DDLS), and retinal nerve fiber layer (RNFL) asymmetry. Results Eighty-one consecutive patients from the Wills Eye Hospital glaucoma service were enrolled, 60 with glaucoma and 21 with ocular hypertension or glaucoma suspect. Thirty-one percent of subjects had MD asymmetry45 dB, 19.7% had CDR asymmetry ≥ 0.20, 26.7% had DDLS asymmetry ≥ 2, and 38.2% had RNFL asymmetry410 microns. Sensitivity values for pupillography were 93.3% (95% CI, 68.1-99.8) for detecting MD asymmetry, 80.0% (95% CI, 51.9-95.7) for CDR asymmetry, 100.0% (95% CI, 73.5-100.0) for DDLS asymmetry, and 69.2% (95% CI, 38.6-90.9) for RNFL asymmetry. Specificity values were 41.2% (95% CI, 24.7-59.3) for detecting MD asymmetry, 32.8% (95% CI, 21.3-46.0) for CDR asymmetry, 33.3% (95% CI, 18.0-51.8) for DDLS asymmetry, and 42.9% (95% CI, 21.8-66.0) for RNFL asymmetry.Pupillography amplitude score was correlated with MD asymmetry (r 2 = 0.41, Po0.001) and area under the curve was 0.84. Conclusion Automated pupillography had higher sensitivity and lower specificity in detecting MD, CDR, DDLS, and RNFL asymmetry. Within the bounds of the cohort tested, this method had limited case-finding ability.