<p>Active-matrix digital microfluidics (AM-DMF) leverages semiconductor-derived electrode arrays to dynamically control thousands of micrometre-scale droplets and has emerged as a transformative platform for high-throughput and precise manipulation of liquid samples. This technology enables various programmable operations, such as droplet generation, transport, mixing and dilution, to be performed with unparalleled accuracy and, thereby, overcomes several limitations of conventional microchannel and passive-matrix digital microfluidics. This Review provides a critical analysis of the design principles and transformative potential of AM-DMF, focusing on its potential biomedical applications in genomics, single-cell analysis and drug discovery. Important contributions of artificial intelligence that increase the efficiency and reliability of complex AM-DMF workflows are also discussed. Despite this considerable progress, further innovation is needed to overcome ongoing challenges such as biofouling, reagent selectivity and electrode stability. This Review outlines future directions for AM-DMF as a versatile tool in life sciences and showcases its role in enabling next-generation droplet manipulation and workflow automation.</p>