Urinary glycosaminoglycans (GAGs) can reflect the health condition of a human being, and the GAGs composition can be directly related to various diseases. In order to effectively utilize such information, a detailed understanding of urinary GAGs in healthy individuals can provide insight into the levels and structures of human urinary GAGs. In this study, urinary GAGs were collected and purified from healthy males and females of adults and young adults. The total creatinine-normalized urinary GAG content, molecular weight distribution, and disaccharide compositions were determined. Using capillary zone electrophoresis (CZE)-mass spectrometry (MS) and CZE-MS/MS relying on negative electron transfer dissociation (NETD), the major components of healthy human urinary GAGs were determined. The structures of ten GAG oligosaccharides representing the majority of human urinary GAGs were determined.