The ability to construct DNA sequencing (DNASeq) libraries from a wide range of starting input masses is essential for many next generation sequencing (NGS) applications. The New England Biolabs® NEBNext Ultra DNA Library Preparation Kit for Illumina (Catalog # E3730) provides users with the ability to construct indexed DNASeq libraries from inputs ranging from 5 ng to 1 ug of starting DNA. In this technical note, we describe the automation of the New England Biolabs NEBNext Ultra DNA Library Preparation Kit for Illumina on the Beckman Coulter Biomek FXP Dual-Arm Multichannel 96 and Span-8 automated liquid handler (Biomek FXP). The automation method allows the user to prepare up to 96 individually indexed DNASeq libraries in approximately 4-1/2 hours. An intuitive HTML-driven user interface allows the user to specify the number of samples to process (up to 96 samples), and which size selection option to be used. The user interface also provides users with the option to utilize either off-deck incubations using an external thermocycler, or to perform incubations on-deck with a Biometra T-Robot thermocycler integrated to the Biomek FXP liquid handler. To help simplify and reduce errors during system setup, an HTML-driven reagent calculator is presented to the user with information on the required reagents, their respective volumes, and their location on the instrument deck based on the user’s input regarding number of samples and steps to be run. The method also incorporates all recommended stop points described in the NEBNext Ultra DNA Library Preparation Kit for Illumina protocol, allowing users the maximum amount of fl exibility in planning their experiments.