The objective of this investigation was to develop a high performance liquid chromatographic method for the determination of Penicillin G,which is widely used in clinical practice. The quantification was carried out using a RP C-18 column in isocratic mode with mobile phase acetonitrile: water: sodium acetate.Cephalexin was used as internal standard. The detection was carried out at 245 nm which was used for direct evaluation of the chromatograms in reflectance/absorbance mode and the good linear relationship was found. The calibration curves were established as dependence of peak height and peak area versus concentration. Standard deviation obtained from calibration curves was compared. Precision and detection limits was validated and found to be satisfactory. The method was found to be reproducible and convenient for quantitative analysis of penicillin G in their raw materials and their dosage forms.