In sprouting angiogenesis, a key process involved in the development and the intravasation of tumor tissues, the growth of vessel sprouts, is determined by migration of single endothelial cells (ECs). This paper presents an on-chip assaying method to investigate the migration of individual ECs by simulating vessel sprouts with microchannels. When chemical stimulus is present, ECs were observed to migrate individually toward the source of factors instead of migrating collectively. The validity of this method is shown by inducing EC migration with glioma cell coculture and culture media doped with vascular endothelial growth factor (VEGF) 165. A positive correlation between cell displacement and VEGF 165 concentration was observed. Difference in migrating ability among cells was reflected by tracking single cells, which could reveal cell heterogeneity in susceptibility to stimulus.