This study describes the application of electrospray ionization mass spectrometry in order to identify ten antibiotics (macrolides, penicillins, aminoglycosides). An optimum procedure was developed for determination of antibiotics of dierent grade. Positive ion spectra of most antibiotics are higher in intensity including an [M+H] + ion and produce less fragmentation and are more informative compared to the negative ion spectra. The group of antibiotics exhibits the same characteristic fragmentation. The data base was developed for identication of antibiotics comparing of their molecular and fragment ions. The results of the study showed that the method with electrospray ionization is simple and quick which is useful in the routine determination of antibiotics and in their pharmaceutical dosage forms.