Ultrasonic standing wave (USW) manipulation is a technology that has been used for separating, sorting, enriching and trapping particles and cells in microfluidic devices including microchannels, microchambers and microwells. One application area is to use the technology for 3D cell cultures on a chip. Such USW-formed 3D cultures have been used for high content screening of tumor spheroids interacting with chemotherapeutic drugs and immune cells. For this purpose, we have developed multiwell microplates designed for high-resolution optimal microscopy. In these microplates, hundreds of tumor spheroids can be formed and shaped by the ultrasound in parallel, followed by high-quality imaging in 3D. However, in our previous work, our USW-based method was not compatible with live cell imaging. Instead, the method was based on active temperature regulation and high-power RF amplification, including bulky and expensive instrumentation. To address this, a novel transducer configuration in combination with an adaptive ultrasonic actuation method has been designed and characterized. The actuation method is applied to a chip-based high-content multi-well screening platform for USW-mediated formation of spheroids. The methodology results in better control of the shape of formed spheroids, eliminates the need for active temperature control and costly RF amplifiers, and enables live-cell microscopy-based imaging during spheroid formation and maturation.