Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an important tool for high-throughput N-glycan profiling and, upon use of tandem MS, for structure determination.By use of MALDI-MS imaging (MSI) in combination with PNGase F treatment, also spatially-correlated N-glycan profiling from tissue sections becomes possible. Here we coupled laser-induced postionization, or MALDI-2, to a trapped ion mobility quadrupole time-of-flight mass spectrometer (timsTOF fleX MALDI-2, Bruker Daltonics).We demonstrate that negative ion mode MALDI-2-MSI is capable of providing high quality N-glycan tissue distributions. Combined with the advantageous fragmentation behavior of [M–H]– ions, exceedingly rich structural information on the composition of complex N-glycans was moreover obtained directly from thin tissue sections of human cerebellum and upon use of low-energy collision-induced dissociation tandem MS. Providing a complete package for MS-based glycan research, MALDI-2 will become a valuable tool in glycobiology research.