PURPOSE: To develop a method to measure the depth profile of microvacuoles (MVs) in intraocular lenses (IOLs) and to characterize, after accelerated aging, the glistening of an acrylic hydrophobic IOL. SETTING: University of Milano-Bicocca, Milan, Italy. DESIGN: In vitro study. METHODS: A heat treatment was applied in vitro to Basis V IOLs exposed to deionized water (24 hours at 45 ± 1°C, rapid cooling, and 24 hours at 24 ± 1°C). Thirty images (area 1.2 mm) of each IOL were acquired by a microscope, focusing on sequential planes every 23 ± 2 μm. By tracking the traces of each MV in consecutive images, the coordinates of the MV centroids along the IOL thickness were construed by an automated procedure, and in the generated single-focus stacked image, MVs were counted by an automated method. RESULTS: MV density was found normally distributed along the IOL depth profile (Jarque-Bera test). In focus-stacked images, the MV automated counting was found accurate within 5% vs manual counting, and MV volume density of the order of 10 mm was estimated. It was observed that stacks of 15 images provided a 4% lower MV volume density compared with the stacking of 30 images. CONCLUSIONS: The assessment of the number of MVs by the acquisition of a single image of an IOL was influenced by the distance of the selected plane from the IOL surface. The decrease in MV density approaching the IOL edges can be explained as a consequence of the diffusion of water toward the external environment after accelerated aging.