RNA-binding proteins (RBPs) are essential regulators of RNA metabolism and gene expression, influencing processes such as splicing, stability, localization, and translation. Despite their critical roles in health and disease, including cancer, identifying RNA–protein interactions remains challenging due to technical limitations and biases of existing methods. Here we review and compare experimental techniques—including in vitro affinity purification, in vivo crosslinking, and proximity labeling—and computational prediction tools for RBP identification. We assess their strengths, limitations, and applicability across biological contexts, emphasizing the benefits of integrating experimental and computational strategies. Our analysis provides practical guidelines for selecting appropriate methodologies tailored to different cell types and research goals. These insights aim to facilitate more accurate mapping of RNA–protein interactomes, thereby advancing understanding of RBP functions and supporting the development of novel therapeutic interventions targeting RNA–protein complexes.

RNA-binding proteins (RBPs) are essential regulators of RNA metabolism and gene expression, influencing processes such as splicing, stability, localization, and translation. Despite their critical roles in health and disease, including cancer, identifying RNA-protein interactions remains challenging due to technical limitations and biases of existing methods. Here we review and compare experimental techniques—including in vitro affinity purification, in vivo cross-linking, and proximity labeling—and computational prediction tools for RBP identification. We assess their strengths, limitations, and applicability across biological contexts, emphasizing the benefits of integrating experimental and computational strategies. Our analysis provides practical guidelines for selecting appropriate methodologies tailored to different cell types and research goals. These insights aim to facilitate more accurate mapping of RNA-protein interactomes, thereby advancing understanding of RBP functions and supporting the development of novel therapeutic interventions targeting RNA-protein complexes.