An approach for rapid HPLC-based profiling for new GABA_A ligands of natural origin has been developed. Active extracts are separated by a single injection of 3 - 10 mg of extract onto a semi-preparative (150 × 10 mm i. d.) HPLC column with gradient elution and time-based fractionation. The microfractions are tested in an automated two-microelectrode voltage-clamp assay on Xenopus oocytes expressing recombinant GABA_A channels composed of α₁, β₂ and γ_2S subunits. The protocol has been validated by spiking experiments with inactive extract and the GABA_A receptor ligand magnolol, and by profiling of active extracts such as valerian extract containing the known GABA_A receptor ligand valerenic acid. For dereplication of GABA containing extracts, we established a rapid and simple procedure by which GABA is analyzed as OPA derivative by reversed-phase HPLC. This dereplication protocol was validated with plant and fungal extracts which had been previously tested active or inactive in the oocyte assay and with spiking experiments.