© 2020 Elsevier B.V.Bisecting N-glycan structures, which are commonly observed in glycoproteins, regulate many important functions in organisms. Tandem mass spectrometry is the most frequently utilized approach for the identification of bisecting N-glycan structures. However, it can be difficult to obtain fragments to recognize bisecting N-glycans based on the analysis performed, i.e., by using hydrophilic interaction liquid chromatography equipped with a fluorescence detector and a quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). The misinterpretation of bisecting N-glycans for other types of N-glycans is possible. Therefore, a method is needed to specifically recognize bisecting N-glycan structures. This report introduces a facile strategy based on tandem mass spectrometry to identify bisecting N-glycans by using a procainamide labeling approach that increases both the mass spectrometric and the fluorescence detection sensitivity of the N-glycans. In this strategy, the precursor ions belonging to bisecting N-glycans were used by extracting the detected diagnostic fragment ions, including proc-H1N3 (m/z 1009.481+) and proc-H1N3F1 (m/z 1155.539+), in the corresponding tandem mass spectra. Subsequently, the structures of the bisecting N-glycans were confirmed. The presented strategy was applied to human IgG glycoprotein and human plasma glycoproteome. Finally, stepping energy-collision induced dissociation (SE-CID) was applied to validate the diagnostic fragments. This approach enables bisecting N-glycans to be verified and can be used for further mass spectrometry-based glycan analysis of biological samples.