Human bronchial epithelial cells (HBECs) are typically cultured at the air-liquid interface (ALI) to functionally recapitulate the human airway. ALI cultures are formed by plating primary HBECs onto porous cell culture inserts and allowing the cells to achieve confluence prior to removing the culture medium from the apical surface of the cells. However, the requirement of porous culture inserts can limit the application of ALI culture to smaller scale experiments, thus largely precluding high-throughput drug screening of differentiated epithelial cultures. Alternatively, HBECs may be cultured and differentiated as spherical aggregates, providing a means for high-throughput study of the differentiated human airway. This protocol describes a method for the culture of differentiated HBECs as spherical structures known as broncospheres or airway organoids.