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The paper studies retention parameters of the therapeutically most frequently used benzodiazepines, i.e. diazepam, oxazepam, medazepam, chlorodiazepoxide, and nitrazepam. The effect of the pH of the mobile phase, percentual representation of methanol, character of the used buffer and the addition of hexanesulfonic acid on the retention time, shape and symmetry of the peaks of the drugs under study were examined. The analysis was carried out on a column filled with Separon SGX C 18. The best results were achieved using the mobile phase methanol-phosphate buffer 0.05 mol/l pH 3.00 65:35, detection with a UV detector at a wavelength of 250 nm. The optimized chromatographic conditions were applied to the analysis of benzodiazepines in rabbit plasma samples.