Antibiotics are extensively used in animal husbandry practices for the treatment of diseases. Four tetracyclines are commercially available as veterinary medicines, of which oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), and doxycycline (DOX). In the present investigation, a novel simple, sensitive, and inexpensive method of analysis was verified for tetracyclines by RP-HPLC system in bulk drug substance and pharmaceutical forms via two stationary phases (C8 & C18). The separation was attained on a 250 mm column length, 4.6 mm internal diameter, 5 µm particle size, C18 and C8 columns with a (55:25:20) 5% glacial acetic acid: acetonitrile: methanolic acid as elution solution at a 355 nm wavelength. The method was performed at a 0.8 ml/min flow rate. This method was applied without an extraction process. The method was validated following ICH and USP guidelines. It was proved to be reproducible and more economical due to the short retention time of less than 6 min by C8 column and 8 min by C18 column which enabled analysis of tetracycline pharmaceutical products with a relatively lower volume of mobile phase. The result of formulation analysis was statistically validated with a high recovery percentage. Furthermore, the developed method was free from the interference of excipients used in the formulation. The validated method was accurate, precise, linear, and reproducible. The data suggest the possibility of using the method in quality control laboratories with better routine analysis of tetracyclines in their dosage forms.