Chronic kidney disease (CKD) is diagnosed and explored through the determination of the glomerular filtration rate (GFR). Our goal was to develop a simple LC-MS method for the determination in serum of 2 popular GFR markers, contrast agents iohexol and iothalamate, for routine use and comparison studies between the two markers. A similar contrast agent, ioversol, was used as an internal standard and the method underwent a rigorous validation protocol based on β-expectation tolerance intervals.We adapted the HPLC-UV method from Cavalier et al. to our LC-MS system. Data treatment for the validation was performed using Multiquant 3.0 (Sciex, Framingham, MA, USA) and e.noval 3.0 software (Arlenda, Liège, Belgium).According to the validation results our method will give accurate and reliable results for concentrations ranging from 6.8 to 250μg/ml for iohexol and 6.15μg/ml to 250μg/ml for iothalamate. In our practice these intervals are sufficient to determine both compounds in most patient samples. Samples with higher detected concentrations can always be diluted into range.With its internal standard and extensive validation, our method is now ready for routine and clinical research use.