The global probiotics market's rapid growth, valued at USD15 billion annually, signifies rising awareness of their health benefits. By 2024, consumption is expected to exceed 12 million tons. The demand is fueled by health awareness and diverse probiotic product availability. Probiotics, generally recognized as safe for consumption, but there are risks associated with less-studied strains or new probiotic strains, genetic mutations and potential contamination by harmful microorganisms.  Thus, it is ultimately important to ensure that the probiotics are safe to consume and meet the quality requirements. However, culture-dependent methods are slow and costly, especially for new strains. This project addresses global demand for rapid, efficient, standardized testing through Next-Generation Sequencing (NGS), offering thorough microbial analysis. The "Sequencing the Future of Probiotics" handbook is the outcome of collaborative efforts by APEC and non-APEC experts in probiotics and NGS. It focuses on NGS-based safety and quality testing. Funded by APEC (SCSC 04 2021A), this project aims to share best practices and recommendations for NGS-based probiotics testing. The handbook consolidates collective expertise into a comprehensive guide.