JuncBASE (Junction-Based Analysis of Splicing Events) is used to identify and classify alternative splicing events from RNA-Seq data. Alternative splicing events are identified from splice junction reads from RNA-Seq read alignments and annotated exon coordinates. In addition to the identification of alternative splicing events. JuncBASE also uses read counts to quantify the relative expression of each isoform and identifies splice events that are significantly differentially expressed across two or more samples. JuncBASE was developed to characterize annotated and novel alternative splicing events throughout Drosophila development as well as splice events that are altered upon knockdown of splice factors. It has been further developed and used for alternative splicing analysis in cancer RNA-Seq studies.