Sequencing analysis finds many applications in various fields of biology from comparative genomics to clinical research. Recent studies, using high-throughput sequencing method, has generated terabytes of data. It is challenging to interpret and draw a meaningful conclusion without the proper understanding of various steps involved in the analysis of such data. This chapter deals with the pipeline to be followed to process the raw RNA sequencing (RNA-Seq) reads, align, assemble, and quantify them in order to draw significant clinical conclusions from them.