Oligosaccharides were covalently labeled with 1-pyrenemethylamine (1-PMA) in 15 min at 75 °C with high yield, separated by quantitative HPLC in less than 20 min, and characterized by off-line Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) mass spectrometry (MS). A new MALDI mass spectral precursor ion for fragmentation studies was observed from 2,5-dihydroxybenzoic acid matrix (DHB, hydroquinone/benzoquinone redox system) through photo-induced reductive elimination. The resulting high-energy protonated glycosylamine provided excellent fragmentation efficiency (Y-, B-ions, and combination losses), with ions covering almost the entire structure of several oligosaccharides at the low picomol level. With the new method, glycans from commercially available standard glycoproteins and glycans from bioengineered β-lactoglobulin expressed in the bgs13 mutant of Pichia pastoris were analyzed.