ErmineJ is software for the analysis of functionally interesting patterns in large gene lists drawn from gene expression profiling data or other high-throughput genomics studies. It can be used by biologists with no bioinformatics background to conduct sophisticated analyses of gene sets with multiple methods. It allows users to assess whether microarray data or other gene lists are enriched for a particular pathway or gene class. This protocol provides steps on how to format data files, determine analysis type, create custom gene sets and perform specific analyses-including overrepresentation analysis, genes score resampling and correlation resampling. ErmineJ differs from other methods in providing a rapid, simple and customizable analysis, including high-level visualization through its graphical user interface and scripting tools through its command-line interface, as well as custom gene sets and a variety of statistical methods. The protocol should take approximately 1 h, including (one-time) installation and setup.