In this study, a simple, sensitive and selective spectroflourimetric method has been developed for the determination of salmon calcitonin (sCT) in ampules. The method is based on the reaction between sCT and fluorescamine at pH 8.5 in borate buffer, resulting in a highly fluorescent derivative. Fluorescence of derivatized sCT solutions was measured by setting the excitation and emission monochromators and slit widths to 390, 484 and 10nm, respectively. Sevaral derivatization parameters were optimized. A calibration graph was constructed using standard solutions of the derivatized calcitonin in the range 0.5-6.0 mu g/mL. Limit of detection and limit of quantification values were determined to be 0.124 and 0.372 mu g/mL, respectively. The proposed method was successfully applied for the determination of sCT in commercially available ampules. High recovery values (101.0-102.0 %), and a low relative standard deviation (RSD %) value (5.3-5.4) proved the accuracy and precision of the proposed method. An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method, as a reference, was also developed for the determination of sCT. A reversed-phase Nucleosil (R) C-18 column (250mm x 4.6mm i.d., 10 mu m particle size, 120 angstrom pore size) was used and the detector was set at 210nm. Statistical comparison of the results of the two methods showed clearly that there was no significant difference between them. Copyright (c) 2012 John Wiley & Sons, Ltd.