Newborn Screening (NBS) has evolved significantly since its inception, yet many treatable rare diseases remain unscreened due to technical limitations. The BabyDetect study used a targeted next-generation sequencing (tNGS) panel to expand NBS to treatable conditions not covered by conventional screening. We present here the analytical validation of this workflow, assessing sensitivity, precision, and reproducibility using dried blood spots from newborns. We implemented strict quality control thresholds for sequencing, coverage, and contamination, ensuring high reliability. Longitudinal monitoring confirmed consistent performance across more than 5,900 samples. Automation of DNA extraction improved scalability, and a panel redesign enhanced the coverage and selection of targeted regions. By focusing on known pathogenic/likely pathogenic variants, we minimized false positives and maintained clinical actionability. Our findings demonstrate that tNGS-based NBS is feasible, accurate, and scalable, addressing critical gaps in current screening programs.