SUMMARY: RNA (ribonucleic acid) molecules have secondary and tertiary structures in vivo which play a crucial role in cellular processes such as the regulation of gene expression, RNA processing and localization. The ability to investigate these structures will enhance our understanding of their function and contribute to the diagnosis and treatment of diseases caused by RNA dysregulation. However, there are no mature pipelines or packages for processing and analyzing complex in vivo RNA structural data. Here, we present rnaCrosslinkOO (RNA Crosslink Object-Oriented), a novel software package for the comprehensive analysis of data derived from the COMRADES (Crosslinking of Matched RNA and Deep Sequencing) method. rnaCrosslinkOO offers a comprehensive pipeline from raw sequencing reads to the identification and comparison of RNA structural features. It includes read processing and alignment, clustering of duplexes, data exploration, folding and comparisons of RNA structures. rnaCrosslinkOO also enables comparisons between conditions, the identification of inter-RNA interactions, and the incorporation of reactivity data to improve structure prediction. AVAILABILITY AND IMPLEMENTATION: rnaCrosslinkOO is freely available to noncommercial users and implemented in R, with the source code and documentation accessible at https://CRAN.R-project.org/package=rnaCrosslinkOO. The software is supported on Linux, macOS, and Windows platforms.