Food authentication is a critical process in verifying the species, origin, and production methods of food products to ensure safety, regulatory compliance, and consumer trust. Traditional methods, such as protein-based assays, often fail in processed or mixed food samples due to protein denaturation. DNA-based techniques, especially DNA barcoding and next-generation sequencing (NGS), offer superior specificity, sensitivity, and robustness. DNA barcoding targets short, standardized genetic regions (e.g., COI, rbcL, ITS) for species identification, while NGS enables high-throughput analysis of complex or multispecies products. These techniques are instrumental in detecting food fraud such as adulteration, mislabeling, and species substitution across a wide range of matrices, including meat, seafood, herbs, spices, and processed foods. This chapter also explores technical challenges like degraded DNA, database reliability, and the need for regulatory validation, as well as emerging solutions such as portable sequencing and AI-driven bioinformatics. Overall, DNA-based authentication is a powerful and evolving tool for ensuring food integrity in modern supply chains.