Iohexol and Iothalamate are nonradioactive contrast reagents, and are used as a marker to determine glomerular filtration rate (GFR). GFR is commonly used to evaluate the overall function of the kidneys as it reflects the rate at which toxins are filtered. It is considered the best index of kidney function. In order to switch from iothalamate to iohexol, a method validation is required to switch protocols. The method being evaluated uses iohexol that is given by bolus IV (single injection), and quantitates the concentration by high performance liquid chromatography (HPLC). HPLC uses high pressure and the affinity of molecules to elute of a column at specific times, called retention times, allowing the the quantitation of iohexol in a sample. Iohexol methodology provides many advantages including using less invasive clinical techniques, needing fewer sampling, and being less expensive than most markers used to determine GFR. The methodology is validated on two Waters® HPLC setups with analysis using EZ Chrom® and EP Evaluator®. EZ Chrom® is the program that stores and analyzes the the information, such as retention times, in order to determine the amount of iohexol. EP Evaluator®, is a program which analyzes data by calculating statistics to determine the precision and accuracy of the results. Assays performed included, inter-assays, intra-assays, analytical sensitivities, linearity, carryover, interferences, analytical comparison and cost analysis. The data showed that within limits, iohexol testing is precise, accurate, and overall valid to implement at Seattle Children’s Hospital in order to determine GFR. This makes it possible for Seattle Children's Hospital to continue to improve patient care by providing a less invasive method to determine GFR.