The Fluorescence Explorer (FLEX) satellite will carry the high-resolution Fluorescence Imaging Spectrometer (FLORIS) that measures the complete fluorescence spectrum emitted by chlorophyll of terrestrial vegetation. This small signal must be validated. One validation approach is comparing the fluorescence signal retrieved from satellite-based measurements with ground based measurements. However, the difference in spatial resolution of the satellite and ground-based instruments and a geolocation mismatch will result in differences in the detected signal and thus, in uncertainties of the validation strategy. In a case study, we identify a representative ground site for validating the fluorescence signal by analyzing surface reflectance measurements from an aeroplane.We define requirements of representativeness for a validation ground site in vegetated areas. Based on those requirements, we identify a suitable position within a case study in central Italy using surface reflectance data from the airborne High-Performance Airborne Imaging Spectrometer (HyPlant) measured in summer 2018. The representativeness is quantified by the relative difference between the single HyPlant pixel representing a ground-based measurement and the averaged signal of several HyPlant pixels that mimics a FLORIS pixel. With this measure, we quantify the validation uncertainty due to spatial resolution and geolocation mismatch. The effect of the temporal evolution of the surface properties on the validation uncertainty due to spatial resolution is investigated.We select the ground site position by minimizing the validation uncertainty due to spatial resolution. Especially for wavelengths larger than 700 nm, this uncertainty is smaller than 2 % for all different reference areas. The largest differences between ground-based like measurement and satellite-like measurement of the surface reflectance is due to geolocation mismatch. The uncertainty due the geolocation mismatch is very large for wavelengths smaller than 720 nm and moderate for wavelengths larger than 720 nm. Thus, the surface reflectance at the chosen position for the validation site is not homogeneous enough for validation purpose. Considering a reference area of 13.5 × 13.5 m2, we quantify temporal stable and small uncertainties for the spectral range between 720 and 800 nm. For an all-embracing validation of the surface reflectance of vegetated areas, the chosen site is not appropriate.