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It takes years to build up a thick layer of nacre in a cultured pearl. The initial fixation and formation of the pearl nucleus, and the growth rate of nacre are important indicators to pearl cultivators. These indicators are traditionally assessed by opening the shellfish for pearl acquisition mechanically causing inevitable waste in aquaculture. Here we report a noninvasive and rapid method based on micro-computed tomography (micro-CT) to evaluate the amount, size, and growth rate of pearls in living freshwater mussel, Hyriopsis schlegelii. Our results show successful detection of overall shapes and amount of pearls through both transmission imaging and 8-s rapid scanning. Accurate dimension-measurements of the cultured pearls were achieved through micro-CT quantitative analysis. This in vivo pearl evaluation did not affect subsequent growth of scanned shellfishes. Our work provides a novel method for quality control in pearl aquaculture.