Updated publication reference for PubMed record(s): 28887379. Glycosylation is among the most abundant and diverse protein post-translational modifications (PTMs) identified to date. The structural analysis of this PTM is challenging due to the diverse monosaccharides which are not conserved among organisms, the branched nature of glycans, their isomeric structures, and heterogeneity in the glycan distribution at a given site. Glycoproteomics experiments have adopted the traditional high-throughput LC-MSn proteomics workflow to analyze site-specific glycosylation. However, comprehensive computational platforms for data analyses are scarce. To address this limitation, we present a comprehensive, open-source, modular software for glycoproteomics data analysis called GlycoPAT (GlycoProteomics Analysis Toolbox). The program introduces ‘SmallGlyPep’ as a minimal linear representation of glycopeptides for MSn data analysis. It enables MS/MS analysis of N- and O-linked glycosylation using a novel scoring scheme to rank the hits based on cross-correlation and probability based analysis. False discovery calculations, parallel computing facilities and user-friendly GUIs (Graphical User Interfaces) are also provided. GlycoPAT is used to analyze site-specific glycosylation on simple glycoproteins, protein mixtures and human plasma cryoprecipitate. The results show that the simultaneous consideration of peptide and glycan fragmentation enables high quality MS spectrum annotation in three common MS/MS fragmentation modes: CID, HCD and ETD.