The cis-iso-α-acids that result from isomerization of hop α-acids, together with their trans-isomers, are the principal bitter components found in beer. Reports indicate cis-isomers are more stable than their trans-isomer counterparts. Therefore, the separation of cis-iso-α-acids could potentially contribute to the production of a more stable beer. Separation was performed using β-cyclodextrin. The optimum complexation conditions that yielded the purest cis-isomer mixture were achieved using water as a solvent at a complexation temperature of 70°C. The ratio of β-cyclodextrin mass to volume of water was 1:8, and the molar ratio of the total amount of iso-α-acids sample to β-cyclodextrin was 1:1. After 2 days of precipitation, the cis-isomer mixture that remained in the supernatant was subjected to ultrafiltration to remove the remaining β-cyclodextrin. The purest trans-iso-α-acids mixture was obtained using the same conditions, except that the molar ratio of the total amount of iso-α-acids sample to β-cyclodextrin was 4:1. The trans-isomers were released from the β-cyclodextrin complex by methanol elution. This method is promising for large-scale production of stereoisomers that can be applied in hopped beverages.