RNA-binding proteins (RBPs) are fundamental regulatory proteins for all forms of transcriptional and posttranscriptional control of gene expression. However, isolating RBPs is technically challenging for investigators. Currently, the most widely used techniques to isolate RBPs are in vitro biochemical approaches. Although these approaches have been useful, they have several limitations. One key limitation to using in vitro biochemical approaches is that RBP–RNA interactions are isolated under nonbiological conditions. Here we review a novel experimental approach to identify RBPs called peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology (Zielinski et al., Proc Natl Acad Sci USA 103:1557–1562, 2006). This technology has two significant advantages over traditional approaches. (1) It overcomes the in vitro limitation of biochemical approaches by allowing investigators to isolate RBP–RNA interactions under in vivo conditions. (2) This technology is highly mRNA specific; it isolates RBPs in an exon-specific manner. By selectively targeting alternatively spliced exons with PAIR technology, investigators can isolate splice variant-specific and mRNA region-specific (5-UTR and 3-UTR) RBP complexes for any mRNA of interest.