A common hallmark of chronic kidney disease (CKD) is kidney fibrosis, which manifests as an increased deposition and turnover of collagens in the kidneys. Current clinical methods of monitoring disease progression in CKD patients do not truly reflect alterations at the tissue level without the use of invasive biopsies. Naturally occurring urinary peptides associated with kidney function and fibrosis have been previously identified using CE-MS as a non-invasive alternative. Moreover, a specific peptide from collagen type III, a highly abundant interstitial collagen, is the target for the C3M enzyme-linked immunosorbent assay (ELISA)-based assay and has been recorded in the CKD273 urinary biomarker panel measured by CE-MS. We aimed to investigate the intensities of the peptides incorporating the C3M sequence captured by CE-MS in urine of patients with CKD and analyze their association with estimated glomerular filtration rate (eGFR) and kidney interstitial fibrosis and tubular atrophy (IFTA). The investigated collagen type III peptides were reduced in abundance in urine of patients with CKD compared to healthy controls and the peptide intensities were independently correlated to eGFR and inversely correlated with IFTA score. Collectively, this analysis supports that peptides containing the C3M sequence are significantly associated with kidney function decline and tissue fibrosis.