Alternative splicing is a process that generates multiple isoforms from a single gene, and its dysregulation is highly linked to diseases. Accurate detection workflow using RNA sequencing remains crucial but challenging, from RNA preparation to data generation and analysis. Solutions addressing these challenges from a data quality control perspective are currently lacking. This review highlights the detection obstacles with potential solutions, recent advancements, and benchmark efforts in isoform and AS detection through RNA sequencing. We further underscore the significance of RNA reference materials, matched datasets, and quality control methods in improving the reliability of transcriptomics for isoform and AS detection.