<p>Protein glycosylation is considered as one of the critical quality attributes which significantly affects the efficacy and safety of biopharmaceutical products. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been widely used to profile glycans formed through glycosylation because it provides the high ionization efficiency for the glycans, typically generating singly charged ions. In this study, we present MALDI-MS-based quantitative glycomics with isotopic Girard’s reagent P labeling of the glycan reducing ends and neutralization of sialic acids for direct comparison of <i>N</i>-glycan profiles from originator and biosimilar products. This method allows us to relatively quantify both neutral and sialylated <i>N</i>-glycans simultaneously. The accuracy, reproducibility, and linearity of the method were evaluated using <i>N</i>-glycans released from human serum IgG. Then, the method was applied to comparative glycan profiling of etanercept originator and its biosimilar, suggesting the applicability of the method to the analytical similarity assessment on glycosylation of biosimilar candidates, especially for early-stage cell line screening.</p>