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CKD is frequently diagnosed only after a significant progression. GFR is the most common indicator of kidney function but is limited in detecting early CKD cases and distinguishing glomerular, tubular, and global CKD. Aiming to provide a glomeruli specific biomarker assay, we developed a peptide immunoaffinity targeted mass spectrometry method for the quantitation of three podocyte specific proteins in human urine: nephrin, podocalyxin, and podocin. Proteins in urine were precipitated, stable isotope labeled peptide standards incorporated, and digested with trypsin. Target peptides were enriched using an online antibody column prior to LC-MS/MS. The performance metrics for nephrin, podocalyxin, and podocin were evaluated: The lower limits of quantitation were 3.8, 22.0, and 5.4 pM, respectively. The intraplate relative error (RE) was within ±10.6%, ± 10.4%, and ±16.1%, and coefficient of variation (CV) was ≤27.2%, ≤ 14.1%, and ≤20.7% accordingly. The interplate RE was within ±7.0%, ± 3.8%, and ±3.0%, and CV was ≤17.2%, ≤ 12.1%, and ≤20.0% for the three analytes. The urinary nephrin, podocalyxin, and podocin concentrations in 60 healthy volunteers and 20 disease samples was measured, thereby establishing the basal levels of these protein and enabling future evaluation of their roles as noninvasive biomarkers of glomerular injury in the clinic.