The expression of different glycans at the cell surface dictates cell interactions with their environment and other cells, being crucial for the cell fate. The development of the central nervous system is associated with tremendous changes in the cell glycome that is tightly regulated. Herein, we have employed biorthogonal Cu-free click chemistry to image temporal distribution of different glycans in live mouse hippocampal neurons during their maturation in vitro. We show development-dependent glycan patterns with increased fucose and decreased mannose expression at the end of the maturation process. We also demonstrate that this approach is biocompatible and does not affect glycan transport although it relies on an administration of modified glycans. The applicability of this strategy to tissue sections unlocks new opportunities to study the glycan dynamics under more complex physiological conditions.