Abstract Summary Spike-in controls are synthetic nucleic-acid sequences that are added to a user’s sample and constitute internal standards for subsequent steps in the next generation sequencing workflow. The Anaquin software toolkit can be used to analyze the performance of spike-in controls at multiple steps during RNA sequencing or genome sequencing analysis, providing useful diagnostic statistics, data visualization and sample normalization. Availability and Implementation The software is implemented in C ++/R and is freely available under BSD license. The source code is available from github.com/student-t/Anaquin, binaries and user manual from www.sequin.xyz/software and R package from bioconductor.org/packages/Anaquin Supplementary information Supplementary data are available at Bioinformatics online.