Herein, we introduce microfluidic superheating as a new method for peptide fragmentation prior to mass spectrometric analysis. The superheating conditions were found to be stable up to 240 °C for more than 30 min without elevated pressure or boiling of the aqueous sample. As proof of principle, we exposed the peptides ACTH1-10 and OVA257-264 to various superheating conditions, causing different degrees of decomposition. Optimized superheating conditions resulted in the entire peptide ladder sequence of the y-ions, allowing the amino acid sequence to be deduced from a single-stage mass spectrum. Thus, obtaining information in the same quality as from tandem mass spectrometry can be achieved by a single superheating step.