This protocol details the steps needed to prepare libraries with unique molecular identifiers (UMIs). It supports a variety of input types including small DNA fragments, such as cell-free DNA (cfDNA), and high molecular weight genomic DNA (gDNA). The workflow generates amplified, indexed libraries with UMI tags for downstream target enrichment and sequencing on Illumina next-generation sequencing (NGS) systems. The products necessary for this workflow include the Twist Library Preparation Kit with Amp Mix, Mechanical Fragmentation and the Twist UMI Adapter System (includes Twist UMI Adapters and Twist Unique Dual Indexed (UDI) Primers). This library preparation protocol is optimized for use with Twist Target Enrichment Kits and should only be performed with reagents specified or their equivalents.