Urine is considered an outstanding biological fluid for biomarker discovery, reflecting both systemic and urogenital physiology. However, analyzing the N-glycome in urine in detail has been challenging due to the low abundance of glycans attached to glycoproteins compared to free oligosaccharides. Therefore, this study aims to thoroughly analyze urinary N-glycome using LC-MS/MS. The N-glycans were released using hydrazine and labeled with 2-aminopyridine (PA), followed by anion-exchange fractionation before LC-MS/MS analysis. A total of 109 N-glycans were identified and quantified, of which 58 were identified and quantified repeatedly in at least 80% of samples and accounted for approximately 85% of the total urinary glycome signal. Interestingly, a comparison between urine and serum N-glycome revealed that approximately 50% of the urinary glycome could originate from the kidney and urinary tract, where they were exclusively identified in urine, while the remaining 50% were common in both. Additionally, a correlation was found between age/sex and the relative abundances of urinary N-glycome, with more age-related changes observed in women than men. The results of this study provide a reference for human urine N-glycome profiling and structural annotations.