Microbiota profiling has emerged as a cornerstone of modern microbiological and biomedical research, offering unprecedented insights into the complex communities of the microorganism inhabiting diverse environments, including the human body, soil and aquatic ecosystems. Recent advancements in sequencing technologies, bioinformatics tools and sample preparation techniques have revolutionized our inability to identify, classify and study microbiota composition and function. This chapter explores the latest techniques for microbiota profiling, including next-generation sequencing (NGS), long-read sequencing, shot gun metagenomics and single-cell approaches. Additionally, we discuss advancements in computational pipelines and artificial intelligence driven analytics that facilitate the interpretation of large-scale microbiota datasets. Challenges such as data reproducibility, standardization and clinical translation are also addressed. This chapter concludes by highlighting the future directions of microbiota profiling and its potential applications in personalized medicine, agriculture and environmental monitoring.