The overarching success of islet transplantation relies on the success in the laboratory to isolate the islets. This chapter focuses on the processes of human islet cell isolation and the ways to optimally provide islet cells for transplantation. The major improvements in regards to the choice of enzyme type, way the digested pancreas tissue is handled to best separate islets from the acinar and surrounding tissues, the various methods of purification of the islets, their subsequent culture and quality assurance to improve outcomes to culminate in safe and effective islet transplantation will be discussed. After decades of improvements, islet cell isolation and transplantation now clearly offer a safe, effective and feasible therapeutic treatment option for an increasing number of patients suffering from type 1 diabetes specifically for those with severe hypoglycaemic unawareness.