Infant biscuits (IBs) are commonly used during the complementary feeding of infants from the 6th month of life. They contain wheat flour and dairy ingredients, which can release the opioid-acting peptides β-casomorphins (BCMs) and gluten exorphins (GEs) after gastrointestinal digestion. In the present study, five model IBs were prepared with or without gluten and different powdered milk derivatives in the formulations. IBs were digested simulating an in vitro static gastrointestinal digestion for infants aged 6-12 months. BCMs and GEs were identified and quantified by UPLC/HR-MS. The amounts of BCM7 and the GE A5 were related to the β-CN and gluten content of the formulations. To date, levels of BCMs and GEs in digests of IBs have not been reported in literature. This work represents an in vitro investigation regarding the release of opioid-acting peptides in IBs. It could add additional knowledge on complementary foods for infant health.