In the last two decades, the development of massive parallel sequencing methods has allowed the sequencing of RNA at an unprecedented resolution, unleashing an enormous wealth of information about the cellular state. Sequencing has accelerated biomedical research by identifying novel mutations, aberrant splicing patterns, splicing isoforms, new gene regulators, and cell-to-cell heterogeneity. In order to efficiently characterize the complexity of the complete transcriptome, there is a steady development for different RNA sequencing [RNA-seq] protocols by improving different steps from library preparation to the data analysis. Furthermore, with the advancements in the sequencing strategies, single-cell RNA sequencing[scRNA-seq] methods have been developed allowing to address the heterogeneity in cell types, and mRNA expression at a remarkable resolution. The majority of these methods involve the conversion of RNA to cDNA and thus amenable to errors, PCR and ligation biases, and inefficiencies of enzymes. Amid these challenges, strategies have been developed to sequence the RNA directly at the single-molecule level which allows to overcome these biases. This chapter provides a brief overview of different sequencing technologies available for the RNA-seq, scRNA-seq and single molecule RNA sequencing along with the different aspects where RNA sequencing has contributed to the biomedical field.