High-throughput sequencing technology is rapidly replacing expression arrays and becoming the standard method for global expression profiling studies. The development of low-cost, rapid sequencing technologies has enabled detailed quantification of gene expression levels, affecting almost every field in the life sciences. In this chapter, we will overview the key points for gene expression analysis using RNA-seq data. First, we will discuss the workflows of RNA-seq data analysis followed by a discussion about the currently available tools for data analysis and a comparison between these tools. The chapter concludes with a discussion about the application of RNA-seq data analysis in livestock. In the appendix, using an example from livestock RNA-seq data, we show a simple script for RNA-seq data analysis.