Fluorescence techniques are widely used as detection methods in a wide range of biological imaging and analytical applications. The purpose of this work is to determine a measurement method which leads to a comparison between different classes of fluorophores in term of stability of the fluorescence signal upon thermal treatment cycles. This kind of investigation can determine whether the fluorophore performance is affected by heating/cooling cycles and to what extent. The fluorophores considered in this work were organic fluorophores belonging to the family of indocyanine dyes (IRIS3 by Cyanine Technologies S.p.A.) in their molecular form or encapsulated within silica nanoparticles, and CdSe/ZnS carboxyl quantum dots (Qdots 565 ITK by Invitrogen). The NIST Standard Reference Material® SRM 1932 fluorescein solution was used in the certified concentration as reference material in order to evaluate the repeatability of the used spectrofluorimeter. The proposed measurement protocol allows to characterize all kind of fluorophores upon thermal treatments. This allows direct comparison of their performance under temperature changes, giving useful guidelines for the selection of the most suitable fluorophore for the envisaged application. Moreover the method appears to be a promising tool for the characterisation of reference fluorescent materials. The experimental results demonstrate that each fluorophore class shows a specific behaviour. The experimental data analysis points out an important hysteresis effect for quantum dots that was not detected for cyanine molecules and was only slightly detected for cyanine doped silica nanoparticles.