Iron (Fe) isotopes are now recognized as useful tracers of Fe sources and biogeochemical processes in natural environments but many uncertainties remain regarding the mechanisms that control their isotopic fractionation. Ultrafiltration techniques applied to separate Fe species could potentially bias Fe isotopic compositions. Here, we investigated frontal centrifugal ultrafiltration. We have set up time-ultrafiltration experiments at low and high Fe concentrations both with and without organic matter (OM) at pH values of 1 and 6.5. The ultrafiltration impact was studied by monitoring the Fe isotope composition in the <30 kDa ultrafiltrates relative to the ultrafiltration time. No Fe isotopes bias resulted from the ultrafiltration technique regardless the Fe and OM concentrations and speciation. This work, therefore, validates the use of the frontal centrifugal ultrafiltration technique to study the signature of Fe isotopes in environmental samples composed of various Fe species sizes such as colloids, nanoparticles, clusters or soluble complexes.