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Technologies for characterizing splicing and isoforms have advanced rapidly, evolving from Sanger sequencing of individual cDNA clones to high-throughput next-generation sequencing of splice junctions, and more recently to long-read sequencing that resolves full-length transcripts at bulk, single-cell, and spatial resolutions. With the growing recognition of their critical roles in human disease, multiple therapeutic modalities have been developed to precisely target splicing and isoform regulation at the DNA, RNA, and protein levels. Clinical-grade small molecules and antisense oligonucleotides that modulate aberrant RNA splicing and isoform switching have become available, offering new hope for previously incurable diseases. 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