Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD) worldwide, but site-specific N-glycosylation signatures underlying DKD renal damage remain poorly characterized. Unlike previous studies relying on suboptimal control tissues, we utilized intact healthy kidney donor (HKD) cortical biopsies as physiologically accurate references. We applied an optimized quantitative glycoproteomic pipeline combining homogenization and non-contact ultrasonic-assisted protein extraction, ZIC-HILIC enrichment, and EThcD-sceHCD-MS/MS to profile intact N-glycopeptides (IGPs) from 20 DKD patients and 20 HKD controls. We identified 1,186 significant differentially expressed IGPs and 111 altered N-glycans derived from 294 glycoproteins in DKD renal tissues (adjusted p < 0.05, FC > 1.5 or < 0.67).