Abstract The use of N-glycan mass spectrometry for clinical diagnostics requires the development of robust high-throughput profiling methods. Still, structural assignment of glycans requires additional information such as MS 2 fragmentation or exoglycosidase digestions. We present a setting which combines a MALDI ionization source with a linear ion trap analyzer. This instrumentation allows automated measurement of samples thanks to the crystal positioning system, combined with MS n sequencing options. 2,5-Dihydroxybenzoic acid, commonly used for the analysis of glycans, failed to produce the required reproducibility due to its non-homogeneous crystallization properties. In contrast, α-cyano-4-hydroxycinnamic acid provided a homogeneous crystallization pattern and reproducibility of the measurements. Using serum N-glycans as a test sample, we focused on the automation of data collection by optimizing the instrument settings. Glycan structures were confirmed by MS 2 analysis. Although sample processing still needs optimization, this method provides a reproducible and high-throughput approach for measurement of N-glycans using a MALDI–linear ion trap instrument.