Portosystemic shunts (PSSs) allow portal blood to bypass the liver and enter the systemic circulation. Definitive diagnosis requires surgical identification, positive contrast portography, ultrasonography, or scintigraphy. This study was designed as a preliminary step to developing an alternative/adjuvant protocol to these imaging modalities. The main goals were to establish a technique for ultrasound-guided percutaneous trans-splenic injection of agitated saline, to evaluate the feasibility of performing the test to explore the postsplenic portal vasculature highlighted by the microbubbles, and to ascertain whether agitated saline microbubbles cross the sinusoidal barrier. Agitated saline was injected into the spleen of 20 healthy sedated dogs under sonographic guidance. The transducer was then repositioned to visualize the portal vein, the caudal vena cava, and the right atrium through different acoustic windows. Satisfactory results were achieved in all dogs. The microbubbles were visualized in all dogs as small intense echo signals within the portal vein at the level of the porta hepatis immediately after injection. In 18 out of 20 dogs, the echogenic signal of the microbubbles disappeared immediately once within the hepatic parenchyma, whereas in two dogs, the echoes from the microbubbles lasted for several seconds within the intrahepatic portal vasculature. The absence of microbubbles beyond the sinusoidal barrier in all of the healthy dogs included in this study makes trans-splenic injection of agitated saline a candidate as an adjuvant technique for the diagnosis of PSS, being easy to perform and repeat, as well as safe and technically feasible.