The study compares official spectrophotometric methods for the determination of proline content in
honey – those of the International Honey Commission (IHC) and the Association of Official Analytical
Chemists (AOAC) – with the original Ough method. Results show that the extra time-consuming treatment
stages added by the IHC method with respect to the Ough method are pointless. We demonstrate
that the AOACs method proves to be the best in terms of accuracy and time saving. The optimized waiting
time for the absorbance recording is set at 35 min from the removal of reaction tubes from the boiling
bath used in the sample treatment. The optimized method was validated in the matrix: linearity up to
1800 mg L1, limit of detection 20 mg L1, limit of quantification 61 mg L1. The method was applied
to 43 unifloral honey samples from the Marche region, Italy.