Current laryngeal injection models rely on the transoral route and are suboptimal due to limited view, narrowed working space, and the need to sacrifice animals for investigation of the injectables. In the present study, a novel surgical model for laryngeal intervention therapy utilizing an ultra-high frequency ultrasound imaging system was proposed. Based on this system, we developed a systemic evaluation approach, from guidance of the injection process, documentation of the injection site of the material, to in vivo longitudinal follow-up on the augmentation and medialization effect by analyzing the ultrasonography data. In vivo animal study. Academic institution. Injection laryngoplasty with hyaluronic acid under ultrasonography guidance was performed on Sprague-Dawley rats one week after induced unilateral vocal paralysis. Ultrasonography was performed at preinjection, immediately postinjection, on Day 2, Day 7 and then weekly for 4 weeks to obtain measurements, including the glottic area, angle between bilateral folds, and vocal fold width ratio. Laryngoscopic and histologic studies were also performed. Unilateral injections to the paralyzed fold were successfully performed as demonstrated by ultrasonographic, laryngoscopic, and histologic studies. The width ratio was significantly increased after injection for 4 weeks, while the glottic airway area was unchanged. Here, a novel surgical model for laryngeal injection utilizing ultrasonography in rats was established. In addition to providing visual guidance for precise localization of the injection, robust documentation of the treatment effect was also demonstrated. This methodology could be beneficial for screening therapeutic agents for treatment of glottic insufficiency.