Notch signaling is essential for the development and homeostasis of multicellular organisms. Dysregulation of Notch signaling leads to several human diseases. Genetic and biochemical studies have shown that O-linked glycosylation on Notch receptors is essential for the transduction of Notch signaling. However, how O-linked glycans regulate the activity of Notch signaling is still unknown. To pursue this, we need to know the whole aspect of glycan modification on Notch. We performed mass spectrometric analysis of Notch1 and 3 to reveal O-glycosylation on each EGF repeat.