Preparation of pure ribosomal subunits carrying lethal mutations is necessary for studying every essential functional region of ribosomal RNA. Affinity purification via a tag, inserted into rRNA proved to be procedure of choice for purification of such ribosomal subunits. Here we describe fast and simple purification method for the 30S ribosomal subunits using affinity chromatography. Streptavidin-binding tag was inserted into functionally neutral helix 33a of the 16 S rRNA from Escherichia coli. Tagged ribosomal subunits were shown to be expressed in E. coli and could be purified. Purified subunits with affinity tag behave similarly to the wild type subunits in association with the 50S subunits, toe-printing and tRNA binding assays. Tagged 30S subunits could support cell growth in the strain lacking wild type 30S subunits and only marginally change the growth rate of bacteria. The presented purification method is thus suitable for further use in purification of 30S subunits carrying any lethal mutations.