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Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. In other words, urine is likely to be a better biomarker source than blood. However, the urinary proteome are affected by many factors. In this study, the effects of three commonly used diuretics (furosemide, hydrochlorothiazide and spirolactone ) on the urinary proteome were analyzed in rats. Urine samples were collected before and after the intragastric administration of diuretics at therapeutic doses and analyzed using LC-MS/MS. Based on quantification by Progenesis LC-MS software, there are 7, 5 and 2 proteins with the p value ≤0.05, a fold change ≥2, a spectral count ≥5 and FDR ≤1%, respectively. Most their human orthologs were considered to be stable in the healthy human urinary proteome. 10 of the 14 proteins have been reported as disease biomarkers in previous studies. So the effects of diuretics should be given more attention in future urinary protein biomarkers studies. The effects of diuretics on urinary proteome are different which can provide clues to elucidate the mechanisms of the diuretics.