Iris transillumination defects may be important markers of disease severity and/or disease activity in patients with pigmentary glaucoma. We present a simple method for quantitation of iris transillumination defects using infrared iris transillumination videography and computerized image analysis. Three observers independently made multiple measurements of percent iris transillumination (the percentage of visible iris that transilluminates) from three standard images using the technique. The interobserver coefficient of variation was 20.35, 6.55, and 8.01%, respectively, for mild, moderate, and marked iris transillumination. The intraobserver coefficient of variation was much smaller for each image, measuring 4.11, 3.23, and 2.38%, respectively, for mild, moderate, and marked iris transillumination. To exemplify the utility of the technique, a single observer measured percent iris transillumination for both eyes of a group of 13 consecutive patients with asymmetric pigmentary glaucoma. There was a significant correlation between the amount of iris trapsillumination and the severity of glaucoma in the eyes of these patients.