A high performance liquid chromatographic method for isocratic mode of elution has been developed for the determination of ampicillin in human plasma. The method comprises injection of the plasma after protein precipitation, separation by reversed-phase C18 column, using heptane sulphonic acid, sodium salt monohydrate as an ion-pairing agent in the mobile phase which consists of pH 3.5 phosphate buffer-methanol. The method involves UV-detection at 225 nm. This method provides a simple tool for the rapid analysis of ampicillin with high degree of accuracy and precision, where the drug level is in microgram range.