Conventional methods for studying paracrine signaling in vitro may not be sensitive to short-range effects resulting from signal dilution or decay. We employ a microfabricated culture substrate to maintain two cell populations in microscale proximity. Individual populations can be quickly retrieved for cell-specific readouts by standard high-throughput assays. We show that this platform is sensitive to short-range interactions that are not detectable by common methods such as conditioned media transfer or porous cell culture inserts, as revealed by gene expression changes in a tumor-stromal crosstalk model. In addition, we are able to detect population-specific gene expression changes that would have been masked in mixed co-cultures. We thus demonstrate a tool for investigating an important class of intercellular communication that may be overlooked in conventional biological studies.