In recent decades, goals aiming to increase gender equality have been widely adopted in global policy-making. This has created a demand for specialized knowledge and evidence to support the design and implementation of gender equality policies. At the international level, multiple initiatives to produce pertinent knowledge that can strengthen gender equality have emerged, resulting in the production of so-called “gender expertise”. In this paper, we focus on a specific knowledge production initiative organized by the World Bank, the Gender Innovation Laboratories (GILs). While research has examined the position of gender experts and the content of gender expertise in international governance, it has overlooked how knowledge about gender relations is produced, and how this contributes to render gender known as a distinct object of knowledge and governance. In this paper, we draw on a practice-theoretical approach – assemblage thinking – to study the practical work mobilized in the GILs to produce, maintain and disseminate knowledge about gender relations. Drawing on interviews with lab researchers and on documents and online-material, we show how the production of gender expertise is dependent on the creation of suitable conditions for knowledge production, the translation of knowledge production into appropriate methodologies, and the packaging of knowledge in fixed dissemination formats. Overall, this paper seeks to contribute to the practice-theoretical literature on knowledge production as well as to discussions in feminist literature on the effects of the use of gender expertise in international governance.