We report a non-viral gene transfer method using ultrasound induced microbubble destruction to allow the uptake of plasmid gene transfer vectors to the cells of the mouse salivary gland. The Luciferase (Luc) reporter gene, driven by a cytomegalovirus (CMV) promoter, was delivered unilaterally to the submandibular salivary gland via retroductal cannulation and Luc expression was monitored with in vivo imaging. The CMV-Luc plasmid was delivered to the salivary gland in a carrier solution containing microbubbles composed of lipid-encased perfluoropropane gas, with two different concentrations of microbubbles used (100 and 15% volume/volume). An Adenoviral (Ad) vector using an identical CMV-Luc expression cassette was used as a positive control at two different dosages. Whereas ultrasound-assisted gene transfer (UAGT) with 100% microbubbles was weak and rapidly extinguished, UAGT with the 15% microbubble solution was robust and stable for 28 days. UAGT seems to be a practicable and promising method for non-viral gene delivery to the salivary glands.