Spironolactone is a potassium sparing diuretic used for decades. Until now, pharmacokinetic (PK) studies of spironolactone have not been conducted in infants and therefore pediatric dosing is based on expert opinion. We aimed to describe the PK profiles of spironolactone and its main metabolites (7alpha-thiomethylspironolactone (TMS) and canrenone (CAN)) in infants up to two years of age. The PK of spironolactone and its main metabolites were evaluated following an oral administration of spironolactone (1mg/kg/dose) to pediatric patients with chronic heart failure, ascites, and/or oedema. The plasma concentration of spironolactone and metabolites (TMS and CAN) was determined using an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Based on rich sampling PK data, the estimation of population PK parameters was performed using nonlinear mixed-effects modelling software Monolix 2018R2. A total of 150 spironolactone, 158 TMS, and 158 CAN concentrations from 23 patients (ages: 3days-21months; median weight 4.3kg (2.2-12.6)) were available for PK analysis. A one-compartment model for spironolactone, TMS, and CAN best fitted the data. The median (range) of individual estimated apparent clearance values were 47.7 (11.9-138.1) L/h for spironolactone, 9.7 (1.5-66.9) L/h for TMS, and 1.0 (0.2-5.9) L/h for CAN. The disposition of spironolactone and metabolites was mainly affected by size of the patient: body weight explained 22% of inter-individual variability of spironolactone clearance. None of the undesirable effects of spironolactone was documented during the study period. The pharmacokinetics of spironolactone and its metabolites was highly variable between patients below 2years of age. Body weight explained a significant part of this variability; this highlights the need to take it into account for dosing prescription in this population. (Clinical trial Registration Number 2013-001189-40).