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Summary: Microbubbles are currently approved for diagnostic ultrasound imaging and are under evaluation in therapeutic protocols. Here, we present a protocol for in vitro sonoporation validation using non-targeted microbubbles for gene delivery. We describe steps for computational simulation, experimental calibration, reagent preparation, ultrasound treatment, validation, and gene expression analysis. This protocol uses approved diagnostic microbubbles and parameters that are applicable for human use.For complete details on the use and execution of this protocol, please refer to Bez et al. (2017).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.