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, alpha-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.