Recent progress in large-scale nucleic acid analysis technology has revealed the presence of vast numbers of RNA species in cells, and extensive processing. To investigate the functions of these transcripts highly efficient methods are needed to analyze their interactions with RNA-binding proteins (RNBPs), and to understand the binding mechanisms. Many methods have been described to identify RNBPs, but none are wholly satisfactory, in part because RNAs are flexible macromolecules that adopt multiple conformations only some of which might bind to specific proteins. Here we describe a novel in vitro RNA-pull-down assay using tRNA scaffolded Streptavidin Aptamer (tRSA), to identify transcript specific RNA binding protein from mammalian cell lysates. The tRNA scaffold functions to stabilize the structure of the aptamer and the attached RNA, increasing the efficiency of the affinity purification.