The study introduces eCLIP, an enhanced CLIP method that improves the identification of RNA-binding protein (RBP) binding sites. eCLIP reduces the required amplification by ~1,000-fold, decreasing discarded PCR duplicate reads by ~60% while maintaining single-nucleotide resolution. It simplifies the generation of paired IgG and size-matched input controls, improving specificity in identifying authentic binding sites. The method was tested on 73 diverse RBPs in HepG2 and K562 cells, demonstrating its ability to enable large-scale and robust profiling with amplification and sample requirements similar to those of ChIP-seq. eCLIP allows for integrative analysis of diverse RBPs to reveal factor-specific profiles and common artifacts in CLIP and RNA-centric perspectives on RBP activity. RBPs play essential roles in regulating gene expression, controlling RNA processing, trafficking, and translation. Defects in RBP function are associated with various genetic and somatic disorders. Technologies like RNA immunoprecipitation (RIP) and CLIP are widely used to identify RNA substrates each RBP interacts with. However, current CLIP methods are technically challenging with high experimental failure rates and low library complexity. eCLIP improves library generation efficiency, saving sequencing costs and enhancing technical and biological reproducibility, enabling RBP binding site identification in limiting samples for low-abundance RBPs and those with few RNA targets. eCLIP was evaluated using the well-characterized RBP RBFOX2, showing significant improvements in library yield and usable read fraction. It demonstrated comparable read density between iCLIP and eCLIP for RBFOX2 binding sites. eCLIP also showed improved signal-to-noise ratio and reproducibility across replicate experiments. The method was used to profile the targets of 132 RBPs in K562 cells and 75 RBPs in HepG2 cells, demonstrating its scalability and reliability. eCLIP enables large-scale in vivo RBP target profiling, with 209 experiments performed for 132 RBPs in K562 and 75 RBPs in HepG2 cells. The method showed high reproducibility and improved efficiency compared to previous CLIP data. eCLIP also enables RNA-centric identification of protein binding to abundant noncoding RNA molecules, revealing specific binding patterns for various RBPs. The study concludes that eCLIP provides a robust, standardized framework for large-scale generation of transcriptome-wide binding maps for RBPs. It maintains single-nucleotide resolution identification of RBP binding sites, dramatically decreases required amplification, and greatly enhances the rate of success at generating libraries with high usable read percentages across diverse RBPs. Additionally, the paired size-matched input controls improve the signal-to-noise ratio for discovery of authentic sites. eCLIP empowers large-scale RBPome-wide profiling efforts, allowing binding site identification with decreased sample requirements andThe study introduces eCLIP, an enhanced CLIP method that improves the identification of RNA-binding protein (RBP) binding sites. eCLIP reduces the required amplification by ~1,000-fold, decreasing discarded PCR duplicate reads by ~60% while maintaining single-nucleotide resolution. It simplifies the generation of paired IgG and size-matched input controls, improving specificity in identifying authentic binding sites. The method was tested on 73 diverse RBPs in HepG2 and K562 cells, demonstrating its ability to enable large-scale and robust profiling with amplification and sample requirements similar to those of ChIP-seq. eCLIP allows for integrative analysis of diverse RBPs to reveal factor-specific profiles and common artifacts in CLIP and RNA-centric perspectives on RBP activity. RBPs play essential roles in regulating gene expression, controlling RNA processing, trafficking, and translation. Defects in RBP function are associated with various genetic and somatic disorders. Technologies like RNA immunoprecipitation (RIP) and CLIP are widely used to identify RNA substrates each RBP interacts with. However, current CLIP methods are technically challenging with high experimental failure rates and low library complexity. eCLIP improves library generation efficiency, saving sequencing costs and enhancing technical and biological reproducibility, enabling RBP binding site identification in limiting samples for low-abundance RBPs and those with few RNA targets. eCLIP was evaluated using the well-characterized RBP RBFOX2, showing significant improvements in library yield and usable read fraction. It demonstrated comparable read density between iCLIP and eCLIP for RBFOX2 binding sites. eCLIP also showed improved signal-to-noise ratio and reproducibility across replicate experiments. The method was used to profile the targets of 132 RBPs in K562 cells and 75 RBPs in HepG2 cells, demonstrating its scalability and reliability. eCLIP enables large-scale in vivo RBP target profiling, with 209 experiments performed for 132 RBPs in K562 and 75 RBPs in HepG2 cells. The method showed high reproducibility and improved efficiency compared to previous CLIP data. eCLIP also enables RNA-centric identification of protein binding to abundant noncoding RNA molecules, revealing specific binding patterns for various RBPs. The study concludes that eCLIP provides a robust, standardized framework for large-scale generation of transcriptome-wide binding maps for RBPs. It maintains single-nucleotide resolution identification of RBP binding sites, dramatically decreases required amplification, and greatly enhances the rate of success at generating libraries with high usable read percentages across diverse RBPs. Additionally, the paired size-matched input controls improve the signal-to-noise ratio for discovery of authentic sites. eCLIP empowers large-scale RBPome-wide profiling efforts, allowing binding site identification with decreased sample requirements and