The last decade has seen an increased interest in the discovery of compounds with bone anabolic activity to treat skeletal disorders such as osteoporosis and increase the well-being of patients. Due to the many technical advantages over classical rodent systems, zebrafish (Danio rerio) has been increasingly used in screening pipelines, in particular those aiming at identifying osteoactive compounds with pharmacological potential. Because compound osteoactivity is mostly determined in zebrafish through the morphometric analysis of bone structures, image analysis, rather than screening assay implementation, molecule availability and image acquisition, represents a bottleneck to the screening throughput. The absence of auto/semi-automatic tools for image analysis of fish bone structures is also a limitation to a broader usage of zebrafish screening pipelines. We present here ZFBONE (for ZEbrafish BONE), an open-source, freely available, user-friendly, rapid and reliable toolset, aiming at accelerating image analysis by automating the morphometric assessment of zebrafish bone structures, but also at increasing data accuracy by reducing operator bias. Tools included in ZFBONE allow users to assess, from 2D images, morphometric parameters of several bone structures (e.g. operculum, caudal fin rays and scales) but also the extent and the intensity of bone-specific colorations. ZFBONE has been developed using the open-source ImageJ software, to make it available to the whole zebrafish research community, but also to have it easily modifiable according to user demands. ZFBONE can also be used toward the standardization of zebrafish screening protocols in academia and industry.Copyright © 2020. Published by Elsevier Inc.