Dravet Syndrome is a genetic epileptic syndrome characterized by severe and intractable seizures associated with cognitive, motor, and behavioral disorders, with onset within the first year of life. Although there are several diagnostic methods such as magnetic resonance imaging (MRI) and electroencephalogram (EGG) to diagnose DS, it is usually difiicult to diagnose in the early stages. In this study, to investigate the metabolic alterations for the onset of Dravet syndrome and for early diagnosis, targeted and non-targeted metabolic profiling were performed in 3D organoid models by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In targeted neurosteroids analysis, the levels of progestins including pregnenolone, progesterone, and 20α-OH progesterone were increased with the differentiation of control organoids, but not in the Dravet syndrome model. In non-targeted analysis, alteration of glycerophospholipids, sphingolipids and fatty alcohols which could affect to microdomain of membrane in brain and play to protect cell from reactive oxygen species were observed. Together with these organoid results, we performed metabolic profiling in brain samples of DS-induced mice, and the results were similar to those in organoids. We found possibility that in vitro experiment with organoids could be replaced an in vivo one, and it expects that our result could contribute to better understanding cause of DS and help to early-diagnose it.