The rapidly increasing number of discovered non-coding RNAs makes the understanding of their structure a key feature toward a deeper comprehension of gene expression regulation. Various enzymatic- and chemically- based approaches have been recently developed to allow whole-genome studies of RNA secondary structures. Several methods have been recently presented that allow high-throughput RNA structure probing (CIRS-seq, Structure-seq, SHAPE-seq, PARS, etc.) and unbiased structural inference of residues within RNAs in their native conformation. We here present an analysis toolkit, named RNA Structure Framework (RSF), which allows fast and fully-automated analysis of high-throughput structure probing data, from data pre-processing to whole-transcriptome RNA structure inference.