RNA molecules participate in virtually all cellular processes ranging from transfer of hereditary information to gene expression control. In cells, many RNAs form specific interactions with proteins often using short nucleotide sequences for protein recognition. Biochemical and structural studies of such RNA-protein complexes demand preparation of short RNAs. Although short RNAs can be synthesized chemically, certain proteins require monophosphate or triphosphate moieties on the 5' end of RNA. Given high cost of chemical triphosphorylation, broad application of such RNAs is impractical. In vitro transcription of RNA by DNA-dependent bacteriophage T7 RNA polymerase provides an alternative option to prepare short RNAs with different phosphorylation states as well as modifications on the 5' terminus. Here we outline the in vitro transcription methodology employed to prepare ≤5-mer oligoribonucleotide for structural and biochemical applications. The chapter describes the principles of construct design, in vitro transcription and RNA purification applied for characterization of a protein that targets the 5' end of RNA.