A rapid, cost-effective spectrofluorometric method was developed for levothyroxine (T4) detection using fluorescein isothiocyanate (FITC) as a fluorescent probe. The method exploits FITC fluorescence quenching by T4 in Tris/phosphate buffers (pH 7.4),with linear ranges of 8.00 × 10⁻⁶ – 6.40 × 10⁻⁵ mol/L and detection limits of 9.60 × 10⁻⁶ mol/L (Tris) and 8.80 × 10⁻⁶ mol/L (phosphate). Stern-Volmer analysis confirmed dynamic q...