Global gene expression analyses in bacteria have undergone a dramatic transformation. Prior to the development of high-throughput sequencing technologies, real-time PCR or microarray studies were the mainstay of assessing differences in gene expression in bacteria. Real-time PCR remains a critical tool for targeted gene expression analyses. However, microarray studies have given way to the plethora of advantages in RNA sequencing (RNA-seq) for the determination of global gene expression (i.e., transcriptome). Increased accessibility to high-throughput sequencing and user-friendly bioinformatics data analysis software have made RNA-seq technology use more widespread. Here, we provide comprehensive methods to perform RNA sequencing of Streptococcus pyogenes strains grown in vitro in standard laboratory media, including cell growth, RNA extraction, ribosomal RNA depletion, and library construction. Considerations for library sequencing and data analysis are also provided.