Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a widely used method in oligosaccharide analysis. Underivatized oligosaccharides are not well-suited for that purpose due to their low ionization efficiency; however, derivatization requires tedious sample purification steps which may lead to sample losses, thereby decreasing its benefit. On-target derivatization performed by the matrix 3-aminoquinoline does not require such purification and yields Schiff bases which can be measured in positive and negative ion mode from one single spot. In negative ion mode, spectra from anionic adducts of the derivatives can be acquired from 1 fmol of oligosaccharide. Furthermore, postsource decay (PSD) fragmentation in positive and negative ion mode is enhanced, providing information on oligosaccharide sequence, linkage, and branching. Optimization of reaction conditions and matrix solution led to a complete and reproducible derivatization for all tested standard oligosaccharides. Finally, the method was applied to trifucosyllacto-N-hexaose and trifucosyl-para-lacto-N-hexaose, two isomers occurring in human breast milk samples, which were easily identified and distinguished.