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We report the first use of ultrasonic standing waves to achieve cell cycle phase synchronization in mammalian cells in a high-throughput and reagent-free manner. The acoustophoretic cell synchronization (ACS) device utilizes volume-dependent acoustic radiation force within a microchannel to selectively purify target cells of desired phase from an asynchronous mixture based on cell cycle dependent fluctuations in size. We show that ultrasonic separation allows for gentle, scalable, and label-free synchronization with high G1 phase synchrony (∼84%) and throughput (3 × 106 cells/h per microchannel).