Acute and chronic kidney diseases are associated with an increased morbidity and mortality. Acute kidney injury (AKI) is a major clinical problem frequently encountered among intensive care unit patients while chronic kidney disease (CKD) affects up to 13% of the general population with high risk of progression to end-stage renal disease. Traditional markers currently used for diagnosis such as serum creatinine, display poor sensitivity and specificity, because they are influenced by several non-renal factors such as age, race and muscle mass. An accurate detection of kidney damage is crucial to enable more appropriate therapeutic intervention to potentially improve the outcome. In the last decade there has been an extensive search for new blood and urinary biomarkers. With the evolution of new technologies, such as proteomics, several candidate protein biomarkers emerged suggesting earlier and more accurate detection of kidney disease than is currently possible with traditional markers.Several of these markers were selected from the literature and a targeted proteomics method able to detect a panel of urinary biomarkers of kidney disease was developed. An online SPE-UPLC-MS/MS (using a triple quadrupole XEVO-TQ-S) was applied. For each protein, between 1 and 4 unique peptide(s) were chosen ending either by a lysine (K) or an arginine (R) in order to be obtained by cleavage using trypsin. MRM transitions of 59 peptides corresponding to 33 biomarkers are followed, with a minimum of 2 mass transitions per peptide and using a 12 minutes chromatographic run time. The 59 corresponding labelled AQUA peptides, containing heavy isotopes of carbon (