The gonadotropin-releasing hormone (GnRH) family includes several hypophysiotropic peptides occupying a central position in the regulatory loop controlling reproduction. Studies are still under way to clarify its biological role and evolutionary implication. Although sequencing of multiple genomes is bringing further advances in the understanding of the evolution of GnRH, there is still a need for biochemical studies aiming to identify GnRH from different species. Using a hybrid quadrupole-time-of-flight (Q-TOF) instrument, a new method for selective and sensitive GnRH detection and characterization from tissue extracts has been developed. The method uses the "precursor ion discovery" mode based on the capability of the Q-TOF analyzer to quickly record alternate mass spectra at low and high collision energy of precursor and product ion spectra, respectively, following liquid chromatographic separation of complex biological mixtures. The method exploits the selective detection of a specific b(2) product ion at m/z 249.1, corresponding to the N-terminus dipeptide pyroglutamic acid-histidine, highly conserved among nearly all species (22 of 24), and deriving from the preferential fragmentation of GnRHs carrying the dipeptide. Importantly, the method also includes acquisition of the product ion spectra from any candidate precursor ion, thereby allowing the determination of sequence information to confirm the GnRH identity or to isolate new ones.