In the paper, conditions of separation 5 β-lactam antibiotics, consist of ampicillin – AMP, penicillin G – PENG, oxacillin – OXA, cloxacillin – CLO, cephalexin – CEP by RP-HPLC are described. The column is Supelcosil ODS (C18), 250mm×4,6mm, 5µm particles. The mobile phase of RP-HPLC is mixture of ACN/MeOH/buffer of 10/20/70 (v/v), pH 3,5.  The limted of detection of  5 β-lactam antibiotics is 0,01 - 0,1 μg/ml. The method determined AMP and CEP in urine of recovery of 86,33% đến 90,62%.