Doshisha Women's College 1 article published in JoVE Neuroscience Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System Takayuki Ueno1,2, Kiyoshi Kawasaki2, Takeo Kubo1 1Department of Biological Sciences, Graduate School of Sciences, The University of Tokyo, 2 Here we describe our detailed protocol for the preparation of single-cohort honeybee colonies – a useful tool for analyzing the role-associated worker physiology. We also describe detailed protocols for treating workers with juvenile hormone and ecdysone to evaluate the involvement of these hormones in the regulation of worker behavior and/or physiology.