Thyroglobulin (Tg) is fundamental for differentiated thyroid cancer (DTC) monitoring. Tg detection can be enhanced using recombinant human thyroid-stimulating hormone (TSH) (rhTSH). This study is aimed to evaluate the use of the rhTSH stimulation test when using a high-sensitivity Tg assay. We retrospectively studied 181 rhTSH tests from 114 patients with DTC and negative for antithyroglobulin antibodies (anti-TgAb). Image studies were performed in all cases. Serum Tg and anti-TgAb were measured using specific immunoassays. rhTSH stimulation in patients with basal serum Tg (b-Tg) concentrations lower than 0.2ng/mL always resulted in rhTSH-stimulated serum Tg (s-Tg) concentrations lower than 1.0ng/mL and negative structural disease. In patients with b-Tg concentration between 0.2 and 1.0ng/mL, s-Tg detected one patient (1/30) who showed biochemical incomplete response. Patients with negative images had lower s-Tg than those with nonspecific or abnormal findings (p<0.05). Receiver operating characteristic curve analysis of the s-Tg to detect altered images showed an area under the curve of 0.763 (p<0.05). With an s-Tg cutoff of 0.85ng/mL, the sensitivity was 100%, decreasing to 96.15% with an s-Tg cutoff of 2ng/mL. Patients with DTC with b-Tg concentrations equal or higher than 0.2ng/mL can benefit from the rhTSH stimulation test.