Glycosylation is one of the most important and ubiquitous posttranslational modifications. To date mass spectrometry (MS) is expected as a powerful tool for determination of these modifications. In particular, matrix assisted laser desorption/ionization (MALDI) has many advantages over other soft ionization methods due to its high sensitivity, simple spectrum, and rapid analysis. However, it is difficult to detect trace amounts of glycopeptides because they show inherent low ionization efficiency compared to unmodified peptides. In order to overcome this problem, we have developed on-plate pyrene derivatization for highly sensitive and easy determination on MALDI-QIT-TOFMS and applied to glycopeptides. Interestingly, the ion yields of glycopeptides were enhanced by on-plate pyrene derivatization, whereas the peptide ion signals drastically decreased in both positive- and negative-ion mode. To elucidate the mechanism in this study, various kinds of peptides were measured after on-plate pyrene derivatization. As the result, generation of positive- and negative-ion from most of peptides used here was suppressed by on-plate pyrene derivatization. The extent of signal suppression seems to be dependent on physicochemical properties of peptides. Especially, more pyrene groups should be introduced to peptides rich in carboxyl groups and the ionization of them was more effectively suppressed even in positive-mode than other peptides. This suggests that the increasing number of pyrene in the peptide molecules have a negative effect on its ion yields. Although the detailed mechanism remains unclear, on-plate pyrene derivatization can selectively enhance ionization of glycopeptides but not peptides and is very useful for analysis of glycoprotains from biological samples. This work was supported by Japan Science and Technology Agency, Development of Technology for Advanced Measurement and Analysis Program.