This toolkit provides practical guidance to governments, funders, civil society organizations and other implementing partners on conducting a gender analysis and using findings to inform HIV prevention, care and treatment programs with key populations. It outlines considerations and steps for conducting a gender analysis; explores how to engage with stakeholders, including key population members, in a meaningful partnership; shares lessons learned from a comprehensive gender analysis in Kenya and an abridged gender analysis in Cameroon; and provides tools and resources for conducting a gender analysis with key populations.