Ribosomes are large protein-RNA complexes involved in translation of mRNA nucleotide sequences into proteins. Multiple ribosomes, polyribosomes (polysomes), bind to a single mRNA in order to initiate translation and protein synthesis. In order to distinguish actively translated RNAs, total polysomes can be isolated from cell lysates and purified by centrifugation through sucrose density gradients. The polysome fraction represents all actively translated cellular RNAs that can be specifically detected using common RNA detection assays, e.g., RT-PCR. The quantity of the target RNA in polysomes fraction indicates its translation state. This chapter provides a protocol for the isolation and fractionation of polysomes from mammalian cell lines.