Streptomyces species, a genus of actinobacteria, are well known for their complex life cycle and excellent potential to produce natural products, which include many clinically and agriculturally important antibiotics. Despite their enormous biotechnological potential, the genetic engineering of Streptomyces remains challenging due to limited genetic tools and the presence of silent biosynthetic gene clusters (BGCs) that require activation for product recovery. Research in genetic engineering and synthetic biology has reached an advanced level, supporting gene mining, strain improvement, strain optimization, and targeted activation of secondary metabolic pathways. Strategies include the development of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing, the design of synthetic genetic components, and the improvement of strain productivity through regulatory genes and enhanced metabolic activity. Moreover, overcoming growth-related limitations in submerged fermentation has further enhanced the industrial applications. These innovations have significantly enhanced the potential of Streptomyces-derived bioactive compounds though strain-dependent variations and construct-specific hurdles persist. The present chapter provides insights into sequencing, bioinformatics, and molecular toolkits that are essential for unlocking the complete biosynthetic capacity of Streptomyces, thereby enabling more efficient antibiotic production and novel drug discovery.