Abstract Introduction: Flumazenil antagonizes the effects of benzodiazepines at γ-aminobutyric acid (GABA) type A receptors in the central nervous system. Flumazenil has been reported to provoke panic attacks in patients with panic disorder (PD) but not in healthy controls. A rapid high-pressure liquid chromatographic (HPLC) method was developed for determination of flumazenil in plasma samples from PD patients receiving flumazenil and the results obtained with that assay are reported here. Methods: Samples from 37 PD subjects receiving 2 mg of flumazenil intravenously were analyzed. Extraction under basic conditions was followed by an HPLC assay with UV detection (250 nm). Lamotrigine was used as internal standard and a standard curve was constructed for each assay run. Flumazenil concentrations were measured in all the subjects in samples collected at 2 and 4 min after the drug administration and in some subjects, measurements were also done in samples collected at 7.5, 15, 30, 45, and 60 min. Results: The procedure was reproducible and linear (from 2.5 to 1000 ng/ml). At 2 and 4 min after flumazenil administration, the concentrations did not differ significantly between panicking and nonpanicking subjects, indicating that the pharmacokinetics of the drug is not the major determinant of the responses. There was a steep decline in the plasma concentration–time profile during the first 4 min, reflecting an extensive and rapid distribution after which the decline was slower. Discussion: The method described here is rapid, replicable, and convenient for the determination of flumazenil in plasma.