Summary Objective  Serum thyroglobulin (Tg) represents a highly specific biomarker for detecting residual thyroid tissue/recurrence/metastases after treatment for differentiated thyroid cancer (DTC). We evaluated the clinical impact of a highly sensitive Tg assay during routine follow‐up of DTC patients. Design  Tg values were measured by a highly sensitive Tg assay during L‐T4 suppressive therapy and after recombinant human thyrotropin (rh‐TSH) stimulation and were compared with those obtained by using a routinely employed Tg assay. Patients  One hundred and sixty consecutive DTC‐treated patients (papillary carcinoma n = 124, follicular carcinoma n = 36) were studied. Measurements  Measured variables included neck ultrasonography, 131I whole body scanning, and Tg assayed by Immulite (Diagnostic Products Corporation, Los Angeles, CA) and by the highly sensitive Access assay (Beckman Coulter, Brea, CA). Results  During L‐T4 therapy, measurable Tg was found in only two patients (1% of total) by Immulite and in 23 patients (14% of total) by Access assay. Using the institutional cut‐off of 2 µg/l after rh‐TSH, a negative response was associated with undetectable Immulite Tg during L‐T4 therapy in all patients (negative predictive value, NPV, 100%) and in 137 out of 152 patients with Access assay (NPV 90%). Measurable Tg during L‐T4 therapy was found in 17% of positive patients with Immulite and in 100% of patients with Access, respectively. Conclusions  The use of a highly sensitive Tg assay may represent a useful diagnostic tool for improving the interpretation of Tg results during monitoring of DTC‐treated patients for the early detection of recurrence and for optimizing the use of the more expensive rh‐TSH test.