This study investigated the possible formation and elimination reactions of 5-hydroxymethylfurfural (HMF) with amino and sulfhydryl groups in commercial biscuits during simulated in vitro gastrointestinal digestion. At the end of gastric phase, significant increase was observed in HMF contents of biscuits (p < 0.05). By high-resolution mass spectrometry (HRMS) analysis, it was confirmed that sugar dehydration products such as 3-deoxyglucosone and 3,4-dideoxyglucosone accumulated in biscuits during baking were converted to HMF under gastric conditions. However, reactions of HMF with amino acids proceeded with the progress of digestion. HRMS analysis in both HMF-amino acid model systems and in biscuits confirmed that formed HMF reacted with amino and sulfhydryl groups through Michael type addition. In addition, formation of Schiff base during intestinal phases led to a significant decrease in the concentrations of HMF (p < 0.05).