AbstractIn this unit, we describe a set of protocols and recommendations for Illumina library preparation. We review best practices in template quantitation methods; template fragmentation methodologies; solid‐phase reverse‐immobilization cleanup, including buffer exchange and size selection; end repair, A‐tailing, and adapter ligation; indexing strategies; considerations regarding whether to use polymerase chain reaction; final library quantification methodologies; and normalization and pooling strategies. These workflows are applicable to both high‐throughput and low‐throughput Illumina library preparation and should help reduce bias, increase cost effectiveness, and produce high library yields. This is an extensive update of the previous version of this unit. © 2019 by John Wiley & Sons, Inc.