Honey bee queens mate in flight with numerous drones. Instrumental insemination (II) is a proven method to control mating, essential for selection, breeding, and research programs. The technique also provides the ability to make novel crosses and to store semen, short term at room temperature and long term cryopreserved. Specialized equipment providing fine precision and a microscope with cold light are required. The basic instrument consists of a sturdy base supporting a set of hooks, queen holder assembly, and micro-manipulated syringe. Featured here are the Schley and Loc instruments and the Harbo syringe. To expose semen for collection, the drone’s endophallus is everted in a two-step process. The semen is skimmed from the endophallus into a syringe, while avoiding the viscous mucus layer. The collected semen is delivered directly into the queen’s median oviduct. Proper positioning of hooks exposes the queen’s vaginal cavity; the valve fold is bypassed, and a measured semen dosage is inserted into the median oviduct. Success, in addition to mastering the technique, requires proper rearing, care, and handling of drones and queens in the preparation and establishment of II queens. Capable of heading full-sized colonies, II queens can be as productive as naturally mated queens.