Rapid separation and quantitative determination of mixtures of amoxicillin, ampicillin, benzylpenicillin, cloxacillin and dicloxacillin was investigated by a reversed-phase high performance liquid chromatographic method with ultraviolet spectrometrical detection at 210 nm.A column Lichrosphere RP-18, 5 p particle size, was used as stationary phase, and mixture of methanol, acetonitrile and aqueous phosphate buffer as mobile phase. Improved retention times were achieved, and all the substances were eluted in 12 minutes. Baseline separation for the three of them, and similar sensitivity of all the five penicillins are the characteristics of the chromatogram. The regression data for the quantitative determination of each penicillin are also given in this work.