Acoustofluidic technologies utilize acoustic waves to manipulate fluids and particles within fluids, all in a contact-free and biocompatible manner. Over the past decade, acoustofluidic technologies have enabled new capabilities in biomedical applications ranging from the precise patterning of heterogeneous cells for tissue engineering to the automated isolation of extracellular vesicles from biofluids for rapid, point-of-care diagnostics. In this Primer, we explain the underlying physical principles governing the design and operation of acoustofluidic technologies and describe the various implementations that have been developed for biomedical applications. We aim to demystify the rapidly growing field of acoustofluidics and provide a unified perspective that will allow end users to choose the acoustofluidic technology that is best suited for their research needs. The experimental set-ups for each type of acoustofluidic device are discussed along with their advantages and limitations. In addition, we review typical types of data that are obtained from acoustofluidic experiments and describe how to model different forces acting on particles within an acoustofluidic device. We also discuss data reproducibility and the need to establish standards for the deposition of data sets within the field. Finally, we provide our perspective on how to optimize device performance and discuss areas of future development.