Aim: Iohexol plasma clearance is used as an indicator of kidney function in clinical and preclinical settings. To investigate the pharmacokinetic profile of iohexol, a rapid, simple method for measurement of iohexol in different matrices and species was needed. Materials & methods: Iohexol was separated on an Accucore C18 column (Thermo Fisher Scientific, CA, USA). Detection was performed on a Thermo Scientific Quantiva tandem quadrupole mass spectrometer. The method was validated according to the requirements for bioanalytical methods issued by the US FDA and European Medicines Agency. Conclusion: We developed and validated a fast and efficient analytical method, suitable for analyzing iohexol in human EDTA plasma, human lithium-heparin plasma, human urine and goat- and pig EDTA plasma, using only one calibration line prepared in human EDTA plasma.