Background: Patients treated for Differentiated Thyroid Cancer (DTC) are followed by thyroglobulin (Tg) testing with standard Tg assays after a stimulation test (Tg-ST), which is expensive and time consuming. High-Sensitivity Thyroglobulin (HS-Tg) assays are purported to replace Tg-ST; acceptable cutoffs however may vary according to assays and patient population. We aimed to evaluate the HS-Tg assay (Roche Elecsys® Tg II) and apply it to clinical use. Method: Analytical evaluations were performed following CLSI standard protocols. Clinical evaluation was done prospectively on 35 DTC patients subjected to Tg measurements both pre- and post- Tg-ST Clinical accuracy performance of HS-Tg was compared to that of conventional Tg-ST protocol utilizing the Siemens Immulite 2000 XPI platform. Results: HS-Tg assay showed an excellent precision (CV=2-3%). The assay reached CV of 11.0% in pooled samples at a mean of 0.048μg/L. HS-Tg results are slightly higher than those from the conventional Siemens Tg assay. HS-Tg ≥ 0.2μg/L showed clinical sensitivity of 1.0 and specificity of 0.81 for predicting recurrence of DTC, which is superior to the Tg-ST protocol using 2μg/L as the cutoff. Two of six patients with HS-Tg results between 0.06-0.2 had a Tg-ST result of >2μg/L, but no recurrence. Conclusions: Although the HS-Tg cut-off of 0.2ug/L is a reliable alternative to Tg-ST in most cases, these tests show divergent results in a proportion of patients making transition from one test to another challenging. Further studies are required to determine the clinical significance of HS-Tg between 0.06-0.2 μg/L.