Production of heterologous proteins in plant systems has become a valuable biotechnological approach as demonstrated by the recent approval of the first plant-made pharmaceutical for the treatment of the rare genetic disorder known as Gaucher’s disease in carrot cell cultures. The main advantages of plants compared to classical expression systems such as bacteria, yeasts or animal cell cultures are the intrinsic safety (e.g. absence of human pathogens), cost-effectiveness and the possibility to manipulate protein post-translational modifications such as glycosylation. Among plant-based production systems, hairy root cultures offer further advantages represented by the possibility of using defined cultivation conditions under contained environment which are attractive for an industrial scale production, the maintenance of product homogeneity and the ease of purification and recovery of the biopharmaceutical product secreted in the culture medium. Several biopharmaceutical products have been successfully produced in hairy root cultures such as vaccine components, enzymes and monoclonal antibodies. In this context, we recently described the production of a tumour-targeting monoclonal antibody with a human-compatible glycosylation pattern in glyco-engineered hairy root cultures. In this chapter we will describe the recent advances in the generation of hairy root cultures expressing heterologous proteins and the strategies adopted to produce biopharmaceuticals, with particular focus on antibodies, and to increase their stability and secretion into the culture medium. Overall, hairy root cultures represent an innovative and promising biotechnological system for the production of plant-made biopharmaceuticals. © Springer Nature Singapore Pte Ltd. 2018.