A gradient RP-HPLC bioanalytical method has been developed for the human pharmacokinetic studies of girisopam, the new 2,3-benzodiazepine compound with anxiolytic effect that has no myorelaxant and anticonvulsive side effects. The compound is an analogue of tofizopam (Grandaxin®, EGIS Pharmaceuticals Ltd., Budapest, Hungary). The method was found to be appropriate for the purposes of human pharmacokinetic studies performed at 25, 50, 100, 200, 325, and 525 mg dose levels. The method allowed the simultaneous determination of girisopam (G) and its four metabolites (4′-hydroxy-G, 7-demethyl-G, 4-hydroxymethyl-G and 4-demethyl-4-oxo-G) identified in previous studies in human plasma. The solutes were separated on Hypersil BDS C18 column and quantified by UV detection at 238 nm. A solid phase extraction (SPE) method using reversed-phase cartridges was developed for sample processing, whereby girisopam and the much more polar metabolites, as well as the internal standard could be extracted in a single step.