Pancreatic cancer cells expressing the surface markers CD133 have been widely reported as cancer stem cells and mainly responsible for tumor recurrence and chemoresistance in pancreatic cancer. In spite of its role as a stem cell marker in pancreatic cancer, its function remains elusive. CD133 (also known as prominin-1) is a pentaspan glycoprotein predominantly localized in lipid rafts, specialized membrane microdomains enriched in crucial signaling proteins. Coexistence of CD133 with these signaling proteins can modulate various signaling pathways that might be responsible for aggressive phenotype of CD133+ cells. This chapter describes a detailed protocol to isolate lipid rafts from CD133+ tumor initiating cells. Purified lipid rafts can be investigated further for protein or lipid composition by mass spectrometry that can shed some light on functional role of CD133 protein in these cancer stem cells.