A comprehensive update on the application of high-throughput fluorescence imaging for novel drug discovery.

introductionHigh-throughput fluorescence imaging (HTFI) is revolutionizing drug discovery by enabling rapid and precise detection of biological targets and cellular processes. Recent advances in fluorescence imaging technologies now provide unprecedented sensitivity, resolution, and throughput. Integration of artificial intelligence (AI) and machine learning (ML) into HTFI workflows significantly enhances data processing, aiding in hit identification, pattern recognition, and mechanistic understanding. AREAS COVERED: This review outlines recent technological developments, integration strategies, and emerging applications of HTFI. It emphasizes HTFI's role in phenotypic screening, especially for complex diseases such as cancer, neurodegenerative disorders, and viral infections. Additionally, it highlights advances in 3D culture systems, organoids, and organ-on-a-chip technologies, which facilitate physiologically relevant testing, improved predictive accuracy, and translational potential, alongside innovative molecular probes and biosensors. EXPERT OPINION: Despite its advancements, HTFI faces ongoing challenges, including data standardization, integration with multi-omics approaches, and scalability of advanced models. However, recent progress in organoid and 3D modeling technologies has enhanced the physiological relevance of HTFI assays, complemented by sophisticated AI and ML-driven data analysis techniques.