The Nextera DNA Flex Library Preparation technique is a state-of-the-art approach created to quickly and effectively prepare superior DNA libraries for next-generation sequencing (NGS). This methodology does not require separate procedures for fragmentation and ligation. The method makes use of a transposase-based technique that binds DNA with sequences that act as adapters while also at the same time fragmenting DNA. Because of this protocol’s flexibility, distinct barcodes may be added, allowing samples to be multiplexed for affordable, high-throughput sequencing. Because of its adaptability, quickness, and low input needs, the Nextera DNA Flex approach is appropriate for a variety of DNA samples, including difficult and low-quality samples. This chapter highlights the benefits and salient characteristics of the Nextera DNA Flex Library Preparation procedure, emphasizing its potential uses in many genetic studies.