A technique to detect residual tetracyclines (TCs) in honey by HPLC and derivative UV-Vis spectra is examined in this study. Chromatograms of honey products often experience overlapped peaks and sloping background due to similar retention properties of components or to the presence of impurities. The derivatives provide resolution of overlapped peaks in an UV-Vis spectrum and eventually bring chromatograms that show pure elution behavior of TCs. Calibration models for oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC) are built out of the derivative spectra of standard solutions of honey and their prediction abilities are examined. The results show that the calibration models for OTC, TC and CTC can yield sufficient levels for the determination. The technique reported here may be effectively used for quantitative determination of TCs.