Bioaccessibility is the proportion of a substance that is absorbed by the intestine and can be utilized by the body. It can be estimated by in vitro protocols, simulating the oral, gastric and intestinal digestions. The absorption of a substance depends on the food source and its processing, as some components of the food matrix can hidden it. The ultrasound technology has been proposed as an alternative for the conventional food processing. However, the effect of this technology on carotenoids, one of the most important bioactive components in foods, needs further studies. For that reason, the aim of this work was to measure the in vitro bioaccessibility of lycopene in guava juice processed by ultrasound. The guava juice containing 15% of pulp was processed using ultrasound (121 W/L, 20 kHz, 1.26 cm2 titanium tip) at 25ºC up to 9 minutes. The in vitro bioaccessibility was evaluated by in vitro digestion (oral, gastric and intestinal digestion), with the carotenoid quantification conducted through HPLC-DAD. The ultrasound processing promoted structural changes on the juice, leading to cellular disruption and increasing its physical stability. On the one hand, the lycopene content was slightly reduced after 9 minutes of ultrasound application. On the other hand, the in vitro bioaccessibility raised from 10% to 20% during the same processing. Consequently, although the ultrasound processing can degrade part of the lycopene, their effect on microstructure modification enhances the lycopene in vitro bioaccessibility, demonstrating that this technology has positive impact on the Guava juice nutritional quality.