This chapter describes the detailed protocol for the isolation and purification of islets of Langerhans from rodent pancreas using collagenase digestion. The first step of the process is to separate and isolate the insulin-producing islets of Langerhans from the rest of the pancreas. The pancreas is excised from the animal, trimmed of non-pancreatic tissues before being inflated and chopped into small pieces. The connective tissue is then broken down with a collagenase enzyme solution to selectively digest the bulk of the exocrine tissue while leaving the endocrine islets intact and separated from their surrounding non-islet tissue. Once this process is completed, the islets of Langerhans are separated from the remaining mixture by centrifugation and purified by the means of hand picking. Once isolated, the subsequent islets can be used for a number of experimental processes.