In previous studies, only 1% of cells in the cultured human pancreatic cancer cell line, PANC-1, were capable of invasion through Matrigel® in a transwell invasion assay. This suggested that invasive PANC-1 cells have unique characteristics conferring this phenotype. This was further investigated using a 3D spheroid model of PANC-1, embedded in Matrigel, coupled with IncuCyte® Live Cell imaging and analysis to capture the movement of the distinct invading population in real time. The identified, invasive PANC-1 were collected and metabolically characterized by CE-TOFMS, and their metabolic profile compared with whole-culture PANC-1. (Fujita et al. 2017, Cancer Science). The invasive PANC-1 cells were distinct from those of the whole cultured PANC-1, with demonstrated increased consumption of ATP, assumed activation of ATP-generating pathways, and higher arginine utilization by NOS. Although they had higher oxidative stress, the invading cells were also more resistant to it with greater survival upon exposure to H2O2. A reduction of intracellular GSH by BSO inhibited PANC-1 invasiveness. Taken together, these results provide a unique metabolic profile for this invasive PANC-1 cell population, as compared to control cells. Such methodology is readily available for similar assessments of invasive cell phenotypes.