This note presents IsoEM2 and IsoDE2, new versions with enhanced features and faster runtime of the IsoEM and IsoDE packages for expression level estimation and differential expression. IsoEM2 estimates fragments per kilobase million (FPKM) and transcript per million (TPM) levels for genes and isoforms with confidence intervals through bootstrapping, while IsoDE2 performs differential expression analysis using the bootstrap samples generated by IsoEM2. Both tools are available with a command line interface as well as a graphical user interface (GUI) through wrappers for the Galaxy platform.