The ionotropic GABA _A receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM). This technology allows the automatically screening of compounds in multiple plates without having to label the cells or use special plates. This methodological approach was first validated by screening the GABA _A receptor expressed in HEK cells using a selection of active compounds in agonist, antagonist, and modulator modes. Then, in a second blind screen of a library of 3041 compounds (mostly composed of natural products), 5 compounds having a specific agonist action on the GABA _A receptor were identified. The hits validated from this unbiased screen were the natural products muscimol, neurosteroid alphaxalone, and three compounds belonging to the avermectin family, all known for having an agonistic effect on the GABA _A receptor. The results obtained were exempt from false negatives (structurally similar unassigned hits), and false-positive hits were detected and discarded without the need for performing electrophysiological measurements. The outcome of the screen demonstrates the applicability of our screening by imaging method for the discovery of new chemical structures, particularly regarding chemicals interacting with the ionotropic GABA _A receptor and more generally with any ligand-gated ion channels and transporters.