The in-flow focusing method, introduced herein, enables rapid and straightforward measurements of the whole-channel acoustic energy of acoustic flow-through devices. The method is applied to assess the whole-channel acoustic energy and acoustophoretic efficiency frequency spectra of a bottom-actuated silicon-glass and a side-actuated glass chip. The input variables are the time-independent geometrical shape of the particle trajectories along the manipulation chamber, the channel cross-section geometry, the physical properties of the reference particles and fluid, and the volumetric flowrate. The outputs are the total acoustic energy, the acoustophoretic efficiency, as well as the acoustic energy density distribution along the channel, which provide a comprehensive overview of the performance of acoustically driven lab-on-a-chip setups. The acoustophoretic efficiency is an important parameter that relates the energy dissipation, and therefore the heat development in the device, to the useful acoustic energy in the channel. Unlike the current state-of-the-art methods such as particle image velocimetry, particle tracking velocimetry, and optical trapping, which are typically limited to small channel areas and are time-consuming, the in-flow focusing method can be applied to long channels and enables rapid measurements of the whole-channel acoustic energy. Published by the American Physical Society 2024