Ultrasound is an established therapy method for bone fracture healing, hyperthermia and the ablation of solid tumors. In this new emerging field, ultrasound is further used for microbubble-enhanced drug delivery, gene therapy, sonoporation and thrombolysis. To study selected therapeutic effects in defined experimental conditions, in vitro setups are designed for cell and tissue therapy. However, in vitro studies often lack reproducibility and the successful transfer to other experimental conditions. This is partly because of the uncertainty of the experimental conditions in vitro. In this paper, the ultrasound wave propagation in the most common in vitro ultrasound therapy setups for cell culture wells is analyzed in simulations and verified by hydrophone measurements. The acoustic parameters of the materials used for culture plates and growth media are determined. The appearance and origin of standing waves and ring interference patterns caused by reflections at interfaces is revealed in simulations and measurements. This causes a local maximal pressure amplitude increase by up to the factor of 5. Minor variations of quantities (e.g., growth medium volume variation of 2.56%) increase or decrease the peak rarefaction pressure at a cell layer by the factor of 2. These pressure variations can affect cell therapy results to a large extent. A sealed cell culture well submersed in a water bath provides the best reproducibility and therefore promises transferable therapy results.