Monitoring glycoprotein therapeutics for changes in glycosylation throughout the drug's life cycle is vital, as glycans significantly modulate the stability, biological activity, serum half-life, safety, and immunogenicity. Biopharma companies are increasingly adopting Quality by Design (QbD) frameworks for measuring, optimizing, and controlling drug glycosylation. Permethylation of glycans prior to analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a valuable tool for glycan characterization and for screening of large numbers of samples in QbD drug realization. However, the existing protocols for manual permethylation and liquid-liquid extraction (LLE) steps are labor intensive and are thus not practical for high-throughput (HT) studies. Here we present a glycan permethylation protocol, based on 96-well microplates, that has been developed into a kit suitable for HT work. The workflow is largely automated using a liquid handling robot and includes N-glycan release, enrichment of N-glycans, permethylation, and LLE. The kit has been validated according to industry analytical performance guidelines and applied to characterize biopharmaceutical samples, including IgG4 monoclonal antibodies (mAbs) and recombinant human erythropoietin (rhEPO). The HT permethylation enabled glycan characterization and relative quantitation with minimal side reactions: the MALDI-TOF-MS profiles obtained were in good agreement with hydrophilic liquid interaction chromatography (HILIC) and ultrahigh performance liquid chromatography (UHPLC) data. Automated permethylation and extraction of 96 glycan samples was achieved in less than 5 h and automated data acquisition on MALDI-TOF-MS took on average less than 1 min per sample. This automated and HT glycan preparation and permethylation showed to be convenient, fast, and reliable and can be applied for drug glycan profiling and clinical glycan biomarker studies.