Summary Sequencing of transposon insertion libraries is used to determine the relative fitness of individual mutants at a large scale. However, there is a lack of tools for specifically analyzing data from such experiments with paired sample designs. Here, we introduce CAFE—Coefficient-based Analysis of Fitness by read Enrichment—a software package that can analyze data from paired transposon mutant sequencing experiments, generate fitness coefficients for each gene and condition, and perform appropriate statistical testing on these fitness coefficients. Availability and Implementation CAFE is implemented in Perl and R. The source code is freely available for download under the MIT License from https://github.com/bengtssonpalme/cafe and http://microbiology.se/software/cafe/ Supplementary information Supplementary data are available at Bioinformatics online. The evaluation data can be obtained from https://microbiology.se/sw/cafe/example_data.tgz