The Barcode of Life initiative represents an ambitious effort to develop an identification system for eukaryotic life based on the analysis of sequence diversity in short, standardized gene regions. Work is furthest advanced for members of the animal kingdom. In this case, a target gene region has been selected (cytochrome c oxidase I) and pilot studies have validated its effectiveness in species discovery and identification. Based on these positive results, there is now a growing effort to both gather barcode records on a large-scale for members of this kingdom and to identify target barcode regions for the other kingdoms of eukaryotes. In this chapter, we detail the protocols involved in the assembly of DNA barcode records for members of the animal kingdom, but many of these approaches are of more general application.