A reverse phase high performance liquid chromatographic (HPLC) method to determine Hydrocortisone (HYD), Oxytetracycline (OXY) and Nystain (NYS) has been developed. The separation was made on a LichrCART^Ŗ C18 column using methanol-NaH₂PO₄/Na₂HPO₄ gradient elution and a diode-array detector measuring at the maximum absorption wavelength for each compound. Quantification limits ranging from 0.19 to 0.85 μg L⁻¹ were obtained. The method was applied to synthetic mixtures and pharmaceutical formations. Analyses were made from the stock solutions and by means of standard addition. Both methods showed similar results, and the claimed levels of pharmaceutical preparations were sown to be in agreement with the results from our analytical method.