BACKGROUND: Plasma clearance of iohexol is used to measure glomerular filtration rate, for which a UHPLC-MS/MS analytical method was previously developed. In real-world conditions, samples may be thawed on arrival and sampled in unvalidated matrices, prompting the need for an improved validation. We aim to improve the method for iohexol determination in plasma with enhanced stability testing, optimized calibration curves, and partial validation in additional matrices. METHODS: Stability testing was conducted up to 9 weeks at room temperature, 4°C, 37°C, and at 37°C with daily two-hour exposure to 55°C. Quintuplicate QC samples were analyzed on 3 days, comparing results from eight-point and two-point calibration curves. Matrix comparison was performed on quintuplicate QC samples in serum, heparin plasma, urine, EDTA whole blood, and heparin whole blood. RESULTS: The method improvements were all compliant with the requirements for bioanalytical methods issued by the US FDA and European Medicines Agency. CONCLUSION: Human EDTA plasma samples can be stored up to 9 weeks at room temperature, 4°C, 37°C, and 37°C with 55°C daily temperature spikes. The samples can be analyzed using a two-point calibration curve and are partial validated for serum-, heparin plasma-, urine-, EDTA whole blood-, and lithium whole blood iohexol samples.