The basic characteristics of 5 columns for high performance liquid chromatography (HPLC) and the mass spectra of phytohormones obtained by HPLC-mass spectrometry (LC-MS) were analyzed to use HPLC efficiently and to determine the optimum conditions for phytohormone analysis. In the case of HPLC, when the MeOH concentration (土20%) of the eluent (1 mL/min) was altered, the retention time became stable after 10 min. For 10 authentic standards of phytohormones, the retention time from 30 to 80% MeOH of an eluent was examined in 5 columns. In the octadecylsilane (ODS) type column, the sequence was zeatin (Z) / zeatin-riboside (ZR) → indole-acetic acid (IAA) → trans-abscisic acid (t-ABA) / indole-propionic acid (IPA) → ABA, and GA₃→ GA₇→ GA₄. In LC-MS, authentic standards and labeled ones of both IAA and ABA types showed almost the same changes in the mass spectrum patterns when the drift voltage increased, indicating the points where the fragmentation occurred. Optimum drift voltages for the highest intensity were 60 V for the IAA and ABA types, 40 V for GA₃ and 80 V for GA₄. Values of drift voltages at which molecular ions could be detected were up to 40 V for IAA, 80 V for ABA, 120 V for GA₃ and 140 V for GA₄.