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 (