•iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) informs on protein-RNA interactions across the transcriptome.•We present the complete data analysis to extract reliable information from iCLIP data.•The workflow covers all steps from initial quality control to RBP binding sites.•We explain the specific requirements for iCLIP data and suggest parameter settings.•Normalisation to background signal provides an estimate of binding site strength. Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to reliably detect RBP binding sites from iCLIP data. The workflow covers all steps from the initial quality control of the sequencing reads up to peak calling and quantification of RBP binding. For each tool, we explain the specific requirements for iCLIP data analysis and suggest optimised parameter settings.