Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiple-charged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.