There is increased global interest in radiolabelled biologicals for clinical applications. Influenced by the search for the ‘biological bullet’, a variety of strategies have evolved for radiolabelling biological products with a view to human health application. This publication describes the challenges that the in vivo use of these products brings and provides essentials from in vitro to in vivo validation in human investigations. Additional attention is paid to safety and the effective use of radiolabelled biologicals in a busy clinical setting. The publication will be a useful resource for nuclear medicine physicians, radiologists, radiopharmacists, pharmacologists and other researchers engaged with radiolabelling biologicals for clinical applications.