The biophysical characterization of new therapeutic molecules is important and mandatory to validate their field of application. The necessary information concerning, for instance, efficacy, toxicity, mechanism of action, cellular uptake, or intracellular location can be gathered using both in vitro and in vivo approaches. Towards this end, researchers frequently rely on the use of fluorescence-based techniques, such as confocal microscopy, flow cytometry, and fluorimetry. However, most molecules are not intrinsically fluorescent. They must be conjugated to a fluorophore to be able to be studied using these methods, which may affect their physicochemical/biological properties. Consequently, the results observed may not be entirely accurate. The choice of the most suitable fluorophore is therefore important. In the last decades, the interest in peptide drugs and drug carriers has increased. Due to intense research using fluorescently labelled peptides, we conjugated four commonly used fluorophores (5(6)-carboxyfluorescein - CF; rhodamine B - RhB; quasar 570 - Q570; tide fluor 3 - TF3), to four different selected peptides. The main objective was the evaluation of the effect of the fluorophores on the end results of different assays. Our results show that fluorophore probes influence peptides. The general considerations are: CF (no toxicity; keep peptides′ activity; low fluorescence signal); RhB (high toxicity; increase Imax (%) in all cells, medium fluorescence signal); Q570 (high toxicity; increase Imax (%) in all cells, high fluorescence signal); and TF3 (medium toxicity; increase Imax (%) in cancer cells, high fluorescence signal). Consequently, the choice of the fluorophore must be judicious and based on the specific application. During the design of a biophysical/biological experiment, more attention should be given to the selection of the fluorophore, since it can affect the results reported by a given technique.