Body fluid metabolomics is a large-scale approach allowing exploring the mechanisms that might underlie specific diseases or sensitivity to processed foods, and identifying associated biomarkers for diagnostics or stratification. Over the past decade, the non-celiac gluten sensitivity (NCGS) is more and more self-diagnosed, which makes the gluten-free diet more frequent, without objective clinical criteria. In fact, because of a lack of clinical indicators, NCGS is poorly understood and challenging to diagnose in contrast to celiac disease. Therefore, finding biomarkers associated with this phenotype is critical for an accurate diagnosis and innovative patient management. To understand the relationship between bread digestion mechanisms and the occurrence of NCGS, a recent approach with in vitro investigation was applied to study the overall digestive process of different breads, combining tools from the oral step thanks to the AM2 masticator apparatus, until the end of digestion thanks to a dynamic digester (DIDGI©) mimicking the physiology of the adult gastrointestinal tract "GIT". One objective in this study was to monitor metabolites produced by in vitro digestates using an untargeted metabolomics approach. In this study, we will outline the methodological strategy taken from preparation of the stomach and intestinal digestates, to acquisition, processing, and annotation of the LC-HRMS data. Interestingly, the first results show fluctuations in certain metabolites identified according to the type of bread digested. This reveals the impact of type of bread on the digestibility and allowed us to emphasize the contribution of metabolomic approach for monitoring the metabolites produced by in vitro digestates.