In vertebrates, intercellular communication is largely mediated by connexins, a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders; hence represent innovative therapeutic targets and identifying modulators of Cx-based HCs is of great interest for better understanding their function and defining new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging while the second is based on the quenching of a fluorescent protein, YFPQL, by iodide after iodide uptake. These assays were then used to screen a collection of 2,242 approved drugs and compounds under development. This study led to the identification of 11 new HC blockers, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in CNS disorders with HC impairments.