Molecularly imprinted polymers were prepared in particle forms by crosslinking methacrylic acid (MAA)) using all trans-retinoic acid as a template. The HPLC column packed with the prepared molecular imprinted polymers showed high capability in separation of retinoid derivatives. The column capacity factor and selectivity increased with increasing MAA to template ratio when the incorporated template amount was fixed, as it statistically generated more binding sites between host molecules and template. Molecularly imprinted polymer particles prepared via an emulsion polymerization method were round-shaped and their sizes were more uniformly distributed, but their separation capability was inferior to those obtained by solution polymerization method. It was presumably because the loss of interaction strength between MAA and the template due to hydrogen bonding either between MAA and water or between template and water during the synthesis of molecularly imprinted polymers.