This review details recent developments in nondestructive ultrasound separation techniques that can be used to separate, trap, or fractionate particles or emulsified droplets from bulk phase liquids. Whereas most previous reviews have focused on small scales or specific applications, this review groups different aspects of the acoustic separation technique and directs it at an audience with interests in separation technologies. The process has potential as an attractive alternative to common separation processes such as centrifugation, membrane filtration, sedimentation, or fluorescence activated cell sorting (FACS). The technology can achieve precise, gentle, and label-free separation in a system that involves no moving parts. The fundamental concepts are presented in detail and previous studies covering a range of different applications are reviewed. The challenges and opportunities for addressing large-scale industrial applications are evaluated.