The aim of the present article was to estimate glomerular filtration rate (GFR) using iohexol (IOX) clearance and evaluate the use of limited sampling models to establish an accurate and clinically suitable test in adult horses and donkeys. Iohexol was administered IV as a bolus at the dose of 75.5 mg/kg in eight horses and eight donkeys through the right jugular vein. Samples were collected through the left jugular vein before marker's administration (time 0) and at 15, 30, 60, 90, 120, 180, 240, 360 minutes, and 12 hours after its injection. Iohexol plasmatic concentrations were determined by high-performance liquid chromatography; plasma clearance was determined by use of a one-compartment model corrected by use of the Brochner-Mortensen formula. The nine-point clearance value was considered a reference for the evaluation of simplified methods. Correlation analysis between the glomerular filtration rate values obtained by the nine-point clearance method and the GFR values determined by the application of simplified sample combinations were performed using Pearson test, linear regression analysis, and one-way ANOVA test; P < .05 was considered significant. Plasma clearance of IOX in horses was 2.58 ± 0.21 mL/min/kg and 1.80 ± 0.10 mL/min/kg in donkeys. No significant difference in GFR estimation was noted among the values estimated by simplified models to those by nine-point method. The mean plasma clearance of IOX determined with three-point clearance model was 2.36 ± 0.14 mL/minute/kg in horses and 2.05 ± 0.40 mL/minute/kg in donkeys. This article reported the determination of GFR in horses and donkeys by single IV administration of IOX. Simplified models were evaluated.