RNA binding proteins (RBPs) are key regulators of RNA biology, mediating processes that span from its biogenesis to decay. Regulatory RNAs interact with canonical (biochemically characterized RNA binding domain, RBD) or non-canonical (unknown RBD) RBPs and they assemble into dynamic ribonucleoprotein particles (RNPs) that control the fate of transcripts. Multiple studies have shown the association between RBPs upregulation and cancer severity, but the RBP-mediated regulatory mechanisms in tumorigenesis still need to be deciphered. To dissect mechanisms of RBP-mediated processes, it is pivotal to accurately identify the RNA targets of RBPs, their binding sites and interaction strengths in order to shed light on the pathway they are involved in. UV-crosslinking followed by immunoprecipitation (CLIP) combined with high-throughput RNA sequencing (RNAseq) is currently the state-of-art method in the field. However, CLIPseq and its variations (HITS-CLIP, PAR-CLIP, iCLIP, irCLIP, eCLIP) present technical and biological limitations related to the low UV-crosslinking efficiency, antibody strength and laborious experimental workflow. To overcome these limitations we developed RNA affinity purification followed by sequencing (RAPseq). RAPseq is an efficient, scalable, and sensitive technology that identifies and quantifies RBP-RNA binding events and modalities in any given transcriptome from any species of interest. This method is based on an in vitro binding assay between a recombinantly purified RBP fused to Halotag and total native RNA. The bound RNA molecules are recovered and sequenced on an NGS platform to retrieve information on the RBP targets, the binding site and binding strength. With RAPseq we aim to map the RNA binding landscape of RBPs, provide new evolutionary insights of essential RBPs through cross-species RAPseq assays and assess the effect of disease-frequent point mutations in RBP binding activity. Furthermore, given the versatility of RAPseq, we developed T7RAPseq and co-RAPseq. T7RAPseq allows to interrogate the impact of RNA modifications on RBP binding activity by comparing modification-devoid and native RNA. co-RAPseq enables assessing cooperative and competitive binding of two or more RBPs to RNA at the same time. co-RAPseq is currently the only method that allows profiling of RNA binding at nucleotide resolution of two or more RBPs in one assay. In the era of RNA-based therapeutics investigating the molecular mechanisms underlying tumorigenesis is crucial to select druggable targets. RAPseq and its multiple applications will facilitate new insight on RBP-dependent regulatory processes. Combined with RBP-perturbation and functional assays, this project will reveal RBP-RNA controlled pathways during oncogenesis, crucial to develop specific therapeutic approach with clinical relevance.