The detection of glycans and glycoconjugates has gained increasing attention in biological fields. Traditional mass spectrometry (MS)-based methods for glycoconjugate analysis are challenged with poor intensity when dealing with complex biological samples. We developed a desalting paper spray mass spectrometry (DPS-MS) strategy to overcome the issue of signal suppression of carbohydrates in salted buffer. Glycans and glycoconjugates (i.e., glycopeptides, nucleotide sugars, etc.) in non-volatile buffer (e.g., Tris buffer) can be loaded on the paper substrate from which buffers can be removed by washing with ACN/H2O (90/10 v/v) solution. Glycans or glycoconjugates can then be eluted and spray ionized by adding ACN/H2O/formic acid (FA) (10/90/1 v/v/v) solvent and applying a high voltage (HV) to the paper substrate. This work also showed that DPS-MS is applicable for direct detection of intact glycopeptides and nucleotide sugars as well as determination of glycosylation profiling of antibody, such as NIST monoclonal antibody IgG (NISTmAb). NISTmAb was deglycosylated with PNGase F to release N-linked oligosaccharides. Twenty-six N-linked oligosaccharides were detected by DPS-MS within a 5-minute timeframe without the need for further enrichment or derivatization. This work demonstrates that DPS-MS allows fast and sensitive detection of glycans/oligosaccharides and glycosylated species in complex matrices and has great potential in bioanalysis.