Drug development has been completely transformed by HTS, which uses automated platforms, miniaturized assays, and sophisticated data processing to quickly assess large chemical libraries against a variety of biological targets. From early manual screening to contemporary ultra-high-throughput methods, this chapter describes the development of HTS, placing special emphasis on assay design, chemical library development, and detecting systems including imaging and fluorescence-based technologies. Approaches to phenotypic screening and functional genomics including CRISPR-Cas9, chemical genomics, and RNA interference are emphasized for their utility in identifying new targets and methods of action. Emerging developments including AI integrated microfluidic devices, quantitative HTS, cHTS for multi-component drug evaluation are presented with important obstacles like assay complexity, cost, false positives/negatives, and library limits. The advancement of tailored treatment paradigms, optimization of therapeutic approaches, and acceleration of discovery are all made possible by integration of machine learning, AI, and HTS in field like precision medicine and agriculture.