Abstract DNA methylation is a widely investigated epigenetic mark with important roles in development and disease. High-throughput assays enable genome-scale DNA methylation analysis in large numbers of samples. Here, we describe a new version of our RnBeads software - an R/Bioconductor package that implements start-to-finish analysis workflows for Infinium microarrays and various types of bisulfite sequencing. RnBeads 2.0 (https://rnbeads.org/) provides additional data types and analysis methods, new functionality for interpreting DNA methylation differences, improved usability with a novel graphical user interface, and better use of computational resources. We demonstrate RnBeads 2.0 in four re-runnable use cases focusing on cell differentiation and cancer.