Imaging N-glycan spatial distribution in tissues using mass spectrometry imaging (MSI) is emerging as a promising tool in biological and clinical applications. However, there is currently no high-throughput tool for visualization and molecular annotation of N-glycans in MSI data, which significantly slows down data processing and hampers the applicability of this approach. Here, we present how METASPACE, an open-source cloud engine for molecular annotation of MSI data, can be used to automatically annotate, visualize, analyze, and interpret high-resolution mass spectrometry-based spatial N-glycomics data. METASPACE is an emerging tool in spatial metabolomics, but the lack of compatible glycan databases has limited its application for comprehensive N-glycan annotations from MSI data sets. We created NGlycDB, a public database of N-glycans, by adapting available glycan databases. We demonstrate the applicability of NGlycDB in METASPACE by analyzing MALDI-MSI data from formalin-fixed paraffin-embedded (FFPE) human kidney and mouse lung tissue sections. We added NGlycDB to METASPACE for public use, thus, facilitating applications of MSI in glycobiology.