The enantiomeric separation of methionine by high performance liquid chromatography on various cyclofructan type chiral stationary phases in the polar-organic separation mode was studied. The effects of the mobile phase composition and temperature on the enantioselectivity of the enantiomers were investigated. The enantioselective separation was attained on the isopropylcarbamate cyclofructan 6 chiral stationary phase with methanol/acetonitrile/acetic acid/triethylamine (75/25/0.3/0.2 v/v/v/v) as a mobile phase. The low retention and no separation were observed on the dimethylphenyl carbamate cyclofructan 7 and naphthylethyl carbamate cyclofructan 6 chiral stationary phases. Three types of detection, low-wavelength UV, polarimetric, and circular dichroism, were used. The UV detection limit for enantiomers was 11 μg mL⁻¹ (S/N = 3) and the polarimetric and circular dichroism detection limits were 10 and 8 times lower. Good linearity was observed from 50 to 500 μg mL⁻¹. The proposed method was applied to determine D- and L-methionine in a dietary supplement sample.