<p>Mass spectrometry combined with stable isotope labeling is a powerful technique for detecting disease-related changes in glycosylation patterns and identifying potential biomarkers. However, stable isotope labeling reagents that simultaneously offer high sensitivity, low cost, and stable sialic acid modifications remain scarce. In this study, we developed a convenient and cost-effective microwave-assisted method for synthesizing a stable isotopic quaternary phosphonium hydrazide labeling reagent pair, ¹⁴N/¹⁵N-P₄HZD, for the quantitation difference analysis of <i>N</i>-glycans using HPLC-ESI-HRMS with high sensitivity and convenience. This strategy features high labeling efficiency, excellent reproducibility, and strong linearity (<i>R</i>² = 0.9984) within a dynamic range spanning two orders of magnitude. The reagent pair is compatible with multiple ion source mass spectrometers and front-end chromatographic separation technologies. In particular, it enhances the ionization efficiency of sialylated <i>N</i>-glycans and facilitates their detection. The relative quantification method has been effectively applied to analyze the variations in <i>N</i>-glycomic profiles from two muscular atrophy models induced by simulated microgravity, specifically the C2C12 cell and hindlimb unloading mouse serum. We discover that these variations display characteristic relevance in both models. <i>N</i>-Glycans Man₃GlcNAc₃Fuc₁ and Man₃GlcNAc₄Gal₁Fuc₁Sia₁ exhibit their potential as biomarkers for the early diagnosis of muscular atrophy. The mass spectrometry method based on the ¹⁴N/¹⁵N-P₄HZD reagent pair offers a convenient and feasible strategy for the difference analysis of <i>N</i>-glycomics, demonstrating significant potential for application in the discovery of clinical biomarkers.</p> Graphical Abstract <p></p>