Introduction The uptake of iodine 131 used in the treatment of differentiated thyroid cancers, is a powerful medical indicator. Recent technological developments, as hybrid SPECT/CT machines, dramatically improve the potential in terms of quantification. Regarding these new opportunities, the aim of this work is to evaluate their performances in order to provide helpful medical tool. Material and methods This study has been implemented on a SPECT/CT device (NM/CT 670 General Electric Healthcare) equipped with 5/8” crystal and high-energy collimator, linked to dedicated software for quantification (Dosimetry Toolkit). Two test objects were used (Jaszczak - Data & Spectrum Lawrence Livermore realistic phantom) to calibrate the acquisition and reconstruction processes and to validate the results. Five refillable spheres (1–2–4–8 & 16 ml) and one polymer object-mimicking thyroid have been used to reproduce clinical situations. Results Taking into account that 3D SPECT data have to be segmented, the threshold value must be first determined. Using the data carried out with refillable spheres positioned in the Jaszczak phantom, a value of 0.4 has been determined. Under these conditions the sensitivity coefficient was equal to 12 cps MBq-1 s-1. The maximum difference registered between expected and measured uptake was 25%, when the Jaszczak phantom with spheres was considered. Neither the parameters of reconstruction algorithm (number of subsets with OSEM) nor the position of the radioactive structures inside the phantom seem to have a more significant influence. A single acquisition using the anthropomorphic Livermore phantom highlighted 15% error for a realistic simulation of a thyroid remnant. Conclusion As accuracy ranged around ±20% is usually sufficient to allow monitoring of thyroid cancer, the results obtained in this study seem consistent with medical requirements. This methodology will now be applied to the pulmonary region in the case of isolated and/or spread uptake.