Natural products are an important source of lead compounds for the development of drug substances. Actinomycetes have been valuable especially for the discovery of antibiotics. Increasing occurrence of antibiotic resistance among bacterial pathogens has revived the interest in actinomycete natural product research. Actinobacteria produce a different set of natural products when cultivated on solid growth media compared with submersed culture. Bioactivity assays involving solid media (e.g. agar-plug assays) require manual manipulation of the strains and agar plugs. This is less convenient for the screening of larger strain collections of several hundred or thousand strains. Thus, the aim of this study was to develop a 96-well microplate-based system suitable for the screening of actinomycete strain collections in agar-plug assays. We developed a medium-throughput cultivation and agar-plug assay workflow that allows the convenient inoculation of solid agar plugs with actinomycete spore suspensions from a strain collection, and the transfer of the agar plugs to petri dishes to conduct agar-plug bioactivity assays. The development steps as well as the challenges that were overcome during the development (e.g. system sterility, handling of the agar plugs) are described. We present the results from one exemplary screening campaign targeted to identify compounds inhibiting Agr-based quorum sensing where the workflow was used successfully. We present a novel and convenient workflow to combine agar diffusion assays with microtiter-plate-based cultivation systems in which strains can grow on a solid surface. This workflow facilitates and speeds up the initial medium throughput screening of natural product-producing actinomycete strain collections against monitor strains in agar-plug assays.