Gonadotropin-releasing hormones (GnRH) are neuropeptide hormones that regulate reproduction in vertebrates. Twenty-five unique chordate GnRH isoforms have been identified, all with very similar molecular architecture. Identification and quantification of endogenous GnRH in biological samples is extremely challenging due to the high levels of sequence similarity among these GnRH peptides and complexity of the biological matrices laden with large numbers of other peptides and protein degradation fragments, and due to low levels of GnRH abundance. In this study, three lamprey GnRH (lGnRH-I, -II, and -III) were extracted from sea lamprey brain tissue and plasma samples by solid-phase extraction (SPE) and identified by a high resolution Q-TOF mass spectrometry (MS). A rapid quantitation method was developed and validated to determine the concentrations of these three lGnRHs by using a UPLC coupled tandem MS in positive ESI multiple reaction monitoring (MRM) mode. Luteinizing hormone-release hormone (LHRH, one of the mammalian GnRHs) was used as the internal standard. The developed quantitation method was fully validated for its recovery, matrix effect, linearity, repeatability, precision and accuracy, and storage stability. This method exhibited excellent linearity in a broad concentration range for all three lGnRHs (R(2)>0.99) and limits of detection (LOD; as low as 0.007 ng/mL). Brain and plasma samples from a total of 280 sea lampreys were analyzed with the developed method to investigate the biological relevance of the lGnRH levels. The concentrations of these three lGnRHs were detected at levels of pictogram per microgram brain tissue and milliliter of plasma. The obtained analytical performance parameters and collected data from real biological samples have proven that this is a robust, sensitive, and fully validated LC-MS/MS method to simultaneously quantify three neuropeptide hormones in complex biological matrices.