Multi-component macromolecular complexes such as ribosome complexes are prone to significant heterogeneity within in vitro reconstituted samples. This can make functional and structural studies difficult, especially in the case of transiently assembling complexes. Translation initiation in prokaryotes proceeds through the formation of transient initiation complexes of the ribosome with initiation factors IF1, IF2 and IF3. The formation of the 30S initiation complex (30SIC) is accomplished in the very early phase of translation initiation, before the actual protein synthesis starts. In order to investigate the structure of the complex comprising the 30S ribosomal subunit, mRNA, fMet-tRNAfMet, IF1 and GTP-bound IF2, we have purified the components and characterized the complex formation by filter-binding assays. This was done with the aim of obtaining a high degree of homogeneity of the complex as desirable for structural studies such as by cryo-electron microscopy (cryo-EM). Here we provide a protocol for the purification of the IFs and for the monitoring of their binding activity by nitrocellulose filter-binding assays using a radio-labelled tRNA. This procedure allows investigating the influence of the different protein factors and of the Mg2+ concentration on the formation of the initiation complex in order to optimize the stochiometry of each component.