This study suggests a novel method for determination of the channel height in asymmetrical flow field-flow fractionation (AF4), which can be used for calibration of the channel for hydrodynamic radiusdeterminations. The novel method uses an oil-in-water nanoemulsion together with multi angle lightscattering (MALS) and elution theory to determine channel height from an AF4 experiment. The methodis validated using two orthogonal methods; first, by using standard particle elution experiments and, sec-ondly, by imaging an assembled and carrier liquid filled channel by x-ray computed tomography (XCT). It is concluded that the channel height can be determined with approximately the same accuracy aswith the traditional channel height determination technique. However, the nanoemulsion method canbe used under more challenging conditions than standard particles, as the nanoemulsion remains stablein a wider pH range than the previously used standard particles. Moreover, the novel method is also morecost effective.