An alternative method for simultaneous baseline separation of alpha and beta-acids homologues and isomers in hop by CD-MEKC with UV detection was proposed. The optimized background electrolyte was composed of 30 mmol/L sodium tetraborate solution, 45 mmol/L sodium dodecyl sulfate, 20 mmol/L beta-cyclodextrin and 10% v/v acetonitrile. The instrumental conditions were evaluated by using a 3(3) Box-Benhken experimental design. In order to demonstrate the applicability of the method, 21 hop samples from different varieties were analyzed. The repeatability intra- and interday tests were performed and relative standard deviations lower than 7% for area and migration times were observed. The present method comprehended 8 min analysis time and revealed to be faster and more efficient when compared to previous reports from literature.