Recent studies have made some progress in tracking the conformation of the nascent chains (1-3); however the earliest steps of folding in vivo (while chain synthesis is underway) remain unclear. This is in part due to a lack of experimental methods to produce stable ribosome-nascent chain complexes with uniform length of the nascent chains. Translation mixtures usually contain heterogeneous fraction of ribosomes with various lengths of the peptide chains due to re-initiation of the translation; this hinders the precise assessment of the conformation of the nascent chains. Here, we describe a detailed procedure to produce homogeneous fractions of ribosome-nascent-chain complexes with defined length of the stalled peptides.