We investigated the endogenous peptidomes of spent hemodialysate, urine, and plasma, to shed light on peptide handling in the kidney. Our study was based on the hypothesis that hemodialysis replaces glomerular filtration and aims at gaining first insight into the comparative distribution of the peptidome/proteome in these body fluids. These data are expected to support insight into the underlying biological and physiological processes that take place in the kidney, which may help to advance treatment in advanced-stage chronic kidney disease and detoxification in renal replacement therapies.