This thesis presents studies of microparticle acoustophoresis, a technique for manipulation of particles in microsystems by means of acoustic radiation and streaming forces induced by ultrasound standing waves. The motivation for the studies is to increase the theoretical understanding of microparticle acoustophoresis and to develop methods for future advancement of its use. Throughout the work on this thesis the author and co-workers1 have studied the physics of microparticle acoustophoresis by comparing quantitative measurements to a theoretical framework consisting of existing hydrodynamic and acoustic predictions. The theoretical framework is used to develop methods for in situ determination of acoustic energy density and acoustic properties of microparticles as well as to design a microchip for high-throughput microparticle acoustophoresis. An experimental model system is presented. This system is the center for the theoretical framework, and its underlying assumptions, of the acoustophoretic microparticle motion in a suspending liquid subject to harmonic ultrasound actuation of the surrounding microchannel. Based directly on the governing equations a numerical scheme is set up to study the transient acoustophoretic motion of the microparticles driven by the acoustic radiation force from sound scattered of the particles and the Stokes drag force from the induced acoustic streaming. The numerical scheme is used to predict the acoustophoretic particle motion in the experimental model system and to study the transition in the motion from being dominated by streaminginduced drag to being dominated by radiation forces as function of particle size, channel geometry, and suspending medium. Next, is presented an automated and temperature-controlled platform for reproducible acoustophoresis measurements suitable for direct comparison to the theoretical framework. The platform was used to examine in-plane acoustophoretic particle velocities by micro particle imaging velocimetry. The in-plane particle velocities are analyzed as function of spatial position revealing the complexity of the underlying acoustic resonances. The resonances and hence the acoustic particle motion are investigated to determine the dependency on driving voltage, driving frequency, and resonator temperature. Furthermore, the in-plane velocity measurements form the basis for a systematic investigation of the acoustic radiation- and streaming-induced particle velocities as function of the particle size, the actuation frequency, and the suspending medium. These measurements are compared to theoretical predictions showing good agreement. The acoustophoretic particle motion, and in particular the acoustic streaming, possesses a three-dimensional character. By using the experimental platform in combination with the Astigmatism micro-PTV technique, preliminary results are presented of the out-of-plane acoustophoretic locities displaying close similarities to the analytical and numerical predictions of the theoretical framework. One of the key problems in microchannel acoustophoresis is to measure the absolute size of the acoustic energy density, an important parameter when designing and optimizing acoustophoresis devices. To facilitate this development, a number of methods are presented to in situ determine the acoustic energy densities by means of acoustophoretic particle motion measured either as particle trajectories, particle velocities, or particle depletion in terms of light-intensity. Other key parameters when designing acoustophoresis devices for particle manipulation are the acoustic properties, density and compressibility of the subject particles. For cells these parameters are widely distributed and are seldom known. For this a method is proposed to in situ determine the density and compressibility of a single particle undergoing acoustophoretic motion in an arbitrary acoustic ﬁeld. Finally, the theoretical framework was used as an essential parameter when designing an acoustophoresis microchip allowing for high-throughput particle separation larger than 1 Liter per hour for a single separation chamber. The design is based on previously reported design of acoustophoretic separators but expanded with temperature control allowing for high-power acoustic actuation. 1 See the list of publications, Section 1.3, and the introduction to each chapter, Section 1.2.