Neuropeptide Y (NPY), a central stimulator of food intake and an energy balance controlling hormone, was quantitatively analyzed in developing brains at birth using microflow liquid chromatography (LC) and triple-quadrupole tandem mass spectrometry (MS/MS). We detected and identified endogenous C-terminal glycine-extended NPY (NPY-Gly 1-37) first, an intermediate of NPY before amidation in the mouse brain using high-resolution Fourier-transform Orbitrap MS and MS/MS. NPY-Gly was present in the fetal brain (E16) at almost the same levels as NPY of 1.92 pmol/g-brain tissue. After birth, NPY in postnatal 2-day brains (P2) was elevated drastically at 11.02 pmol/g-brain (p < 0.05 vs E16) and remained at a high level for the first 10 postnatal days, an important period for the formation of the NPY neural circuit in the brain. Immunohistochemistry unexpectedly showed that the localizations of NPY and NPY-Gly in the hypothalamus were completely different: NPY was localized in the arcuate nucleus, whereas NPY-Gly was already located at pars tuberalis during brain development from a fetus to a neonate to a sexual adult.