Infant formula (IF) constitutes the sole source of mineral intake for infants who are only fed IF. The assurance of the amount of minerals declared on the label and the mineral levels and their chemical forms present a major concern related to providing a good amount of nutrients for absorption by these children. Thus, the objectives of this study were: (i) to evaluate several sample preparation methods for minerals in IF; (ii) to validate an analytical method using an ultrasonic bath for simultaneous determination of the Ca, Cu, Fe, K, Mg, Mn, Na, P and Zn contents in IF by ICP OES and (ii) to establish the optimum analytical conditions of the in vitro method to study the dialyzability of these minerals from IF. The ultrasound-assisted method was shown to conform to 'green chemistry principles', being simple, fast and low cost compared with reference methods. The results were similar to those obtained with reference methods (microwave-assisted acid digestion and dry ashing) with regard to selectivity, sensitivity and linearity (r2 > 0.999). The accuracy and the precision were verified using certified reference materials, with recoveries and coefficients of variation ranging from 91 to 105% and from 1.1 to 5.2%, respectively. For in vitro dialyzability, the conditions established in this study allowed including an overnight step between the gastric and gastrointestinal stages (accuracy and precision ranging from 81 to 108% and 0.4 to 6.3%, respectively), contributing to establishing an in vitro digestion method suitable for infant gastrointestinal conditions.