Glycans are molecules that contain multiple monosaccharides linked by glycosidic bonds and often bind to proteins or lipids to form various glycoconjugates. Glycans became more and more complex with evolution and began to acquire significant functional roles crucial for complex organisms. For this reason, it is crucial to include glycan analysis when determining the physiology, pathophysiology, etiology, or diagnosis of some diseases. Glycan analysis is most often performed by glycan derivatization, where the most commonly used method is the binding of fluorophores by a reductive amination reaction, after which the labeled glycans are most often analyzed by UPLC-MS methods. When choosing a fluorophore to label an N-glycan, it is necessary to think about its suitability and properties in the method which will be used. In particular, with the HILIC-UPLC-FLR-ESI-MS method, fluorescence and ionization of fluorophores need to be considered. As glycans themselves do not have a natural chromophore, the intensity of the fluorescence signal comes from the fluorophore. Thus, if the fluorophore fluoresces poorly, then signals of relatively lower intensities will not rise well enough above the baseline and analysis of such glycans may be difficult or impossible. On the other hand, fluorophore significantly contributes to the ionization of glycans, especially if it has functional groups that are easily protonated or deprotonated, depending on the MS mode. For this reason, there is a need to design fluorophores that will contribute more to the fluorescence and ionization of glycans and the need to quantify these contributions to improve glycan analysis methods involving negative MS mode as these methods are more informative than methods involving positive MS mode. For this purpose, an analysis of N-glycans labeled with 11 different fluorophores was performed. The correlation of LogP and pKa of different fluorophores and signal-to-noise intensity ratios in the negative MS mode was then determined. In this way, it was shown that lipophilicity significantly contributes more to signal-to-noise intensity ratios in the mass spectrum than pKa, ie negative charge in the case of negative MS mode. With this in mind, further research and development of fluorophores that will be suitable for glycan analysis in the negative MS mode are proposed.