Sulfation and phosphorylation are important modifications present on the carbohydrate moieties of glycoproteins. Since these acidic functional groups significantly affect the biological properties of glycoproteins, developing efficient and sensitive methods to identify these structures is essential for understanding the structure/function relationship in numerous species. Mass spectrometry is one method used to characterize glycoproteins and detect the presence of these acidic functional groups. However, the applicability of MS analysis is limited in three ways: 1) Acidic glycopeptides ionize inefficiently; 2) They do not produce abundant glycosidic cleavage ions during (-) ESI-MS/MS experiments (so their structures cannot be assigned); and 3) Distinguishing sulfation and phosphorylation is challenging, since these groups are isobaric. Herein, we have developed ion-pairing methods to overcome these difficulties. Sulfated glycopeptides ionize inefficiently and are not detectable by MS assays in the presence of many nonsulfated glycoforms. MS data acquired before and after the addition of the ion-pairing reagent Lys-Lys-Lys (K3) demonstrate that the ion signal of sulfated glycans can be enhanced because the ion-pairing reagent selectively complexes with these acidic functional groups. In addition to enhancing the ion signal of sulfated glycopeptides, the ion-pairing strategy is useful to obtain the structural information about sulfated glycopeptides. The MS/MS data acquired on the ion-pairing complexes in positive ion mode provide detailed information of the sequence, branching, and type of N-linked glycopeptides, compared to the (-) ESI-MS/MS analysis. Finally, the MS/MS data of uncomplexed sulfated or phosphorylated glycans are identical, but the MS/MS data of the phosphorylated and sulfated ion-pairing complexes are quite different. Thus, this approach can be used to successfully identify which species were phosphorylated and which were sulfated in the complex biological mixtures. In conclusion, the ion-pairing method is a single synergistic approach that discriminates sulfation from phosphorylation, enhances sensitivity, and provides structural information for the acidic glycoproteins.