We evaluated the value of serum-free thyroid hormone and thyrotropin (TSH) concentrations in the detection of peripheral hyperthyroidism during thyroxine suppression therapy. A total of 57 patients on a stable thyroxine dose and 70 controls participated in the study. Serum-free thyroxine (FT4), free triiodothyronine (FT3) and TSH were measured by immunoassays based on time-resolved fluorescence (Delfia). The assay for TSH was a modification of a third generation Delfia hTSH Ultra method. The patients were classified into euthyroid and hyperthyroid subgroups based on clinical signs and symptoms (Wayne index). Systolic time intervals (STI) were measured. The Wayne indices were higher among patients than controls (p < 0.0001). The STI results were similar in patients and controls. Only FT4 had the discriminatory power for classifying euthyroid and hyperthyroid patients according to discriminant analyses. The diagnostic value of FT4 was further assessed by calculating the area under the relative operating characteristic (ROC) curve. The area was 0.707 (SE 0.0918), which was significantly different from an area of 0.5, i.e. the area of a test of no value (p = 0.032). In conclusion, a high serum FT4 concentration indicates hyperthyroidism during long-term thyroxine treatment among thyroid carcinoma patients. Although the degree of TSH suppression can now be exactly monitored with new third generation TSH assays, hyperthyroidism cannot be defined using TSH concentration in thyroid carcinoma patients. Therefore, additional serum FT4 concentration assays are needed in the assessment of hyperthyroidism associated with TSH suppression therapy in thyroid carcinoma patients.