RNA sequencing (RNA-Seq) has become the gold standard for transcriptome analysis, making gene expression quantification the core application in molecular biology research. For well-annotated genomes, the standard RNA-Seq workflow relies on a reference genome-based approach. However, RNA-Seq analysis presents different challenges, including complex computational pipelines, the need for bioinformatics skills, and significant computational requirements. The Galaxy platform addresses these challenges by providing an easy-to-use, web-based solution that integrates multiple bioinformatics tools and databases. This enables researchers without extensive bioinformatics expertise to effectively analyze RNA-Seq data. This chapter aims to describe the process and use of the tools required to perform a reference-based RNA-Seq gene expression analysis using the Galaxy public web server.