Ion mobility spectrometry (IMS) separate ionic species during drift time in the electric field tube. The drift time depends on the mass of the ion, its charge state, and its average rotational cross-section. Therefore, IMS is able to distinguish the same mass compounds. However, it is not clear regarding ion separation behavior. The negative ion of flavonol glycoside with hydroxy groups is having multiple ion species in order to have some ionization site. We analyzed the ion mobility and related collision cross section (CCS) of negative ion of flavonoid glycoside. Kaempferol 3-rutinoside and isorhamnetin 3-rutinoside showed two peaks in the IMS driftgram. On the other hand, quercetin 3-rutinoside and myricetin 3-rutinoside gave only single peak. Furthermore, we will discuss a comparison of the calculated CCS values and the experimental CCS (ΩN2) values using the T-wave Ion mobility spectrometer.