Abstract Summary Experimental methods using microRNA/target ligation have recently provided significant insights into microRNA functioning through generation of chimeric (hybrid) RNA sequences. Here, we introduce Hybkit, a Python3 API, and command-line toolkit for analysis of hybrid sequence data in the “hyb” file format to enable customizable evaluation and annotation of hybrid characteristics. The Hybkit API includes a suite of python objects for developing custom analyses of hybrid data as well as miRNA-specific analysis methods, built-in plotting of analysis results, and incorporation of predicted miRNA/target interactions in Vienna format. Availability and implementation Hybkit is provided free and open source under the GNU GPL license at github.com/RenneLab/hybkit and archived on Zenodo (doi.org/10.5281/zenodo.7834299). Hybkit distributions are also provided via PyPI (pypi.org/project/hybkit), Conda (bioconda.github.io/recipes/hybkit/README.html), and Docker (quay.io/repository/biocontainers/hybkit).