Biomarker is the measurable change associated with a physiological or pathophysiolog-ical process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F;hydro-chlorothiazide, H; and spirolactone, S) on the urinary proteome in rats. Urine samples were col-lected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the criteria of P 6 0.05, a fold change P2, a spectral count P5, and false positive rate (FDR) 61%, 14 proteins (seven for F, five for H, and two for S) were identified by Progenesis LC-MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics.