Ballgown is a flexible differential expression analysis R software package specifically designed for RNA-Seq data. It can identify differentially expressed genes, exons, and transcript isoforms at the transcript level. Its standard workflow is: TopHat2 or HISAT2 alignment → StringTie assembly and quantification (add -B parameter to generate Ballgown readable format) → Differential analysis using the Ballgown R package. Ballgown not only provides statistical tests but also offers a wealth of visualization and data exploration functions, helping researchers deeply understand the changes in the transcriptome. This tool is released through the Bioconductor channel and is an important choice for transcript level differential expression analysis.