14-3-3 proteins are adaptor elements in intracellular signaling pathways. Recently, this protein family has been identified as a relevant therapeutic target involved in many human diseases. Therefore, identification of 14-3-3 proteins in biological systems is very important. Two cationic peptide-based probes are reported for the fluorescence detection of 14-3-3 proteins at physiological pH. The design of these probes consists of two symmetric peptidic arms equipped with a guanidiniocarbonyl pyrrole moiety (an arginine mimetic aka GCP), and an environment-sensitive amino-naphthalimide fluorophore as a third arm. These peptide sequences also contain lysine and phenylalanine/tryptophan amino acids for additional charge-charge and hydrophobic interactions. Both probes show high affinity and sensitivity for the 14-3-3 family, as well as good selectivity against other relevant biological proteins and ions.