Despite considerable research, effective and safe treatments for human pain disorders remain elusive. Understanding the biology of different human pain conditions and researching effective treatments continue to be dominated by animal models, some of which are of limited value. British and European legislation demands that non-animal approaches should be considered before embarking on research using experimental animals. Recent scientific and technical developments, particularly in human neuroimaging, offer the potential to replace some animal procedures in the study of human pain. A group of pain research experts from academia and industry met with the aim of exploring creatively the tools, strategies and challenges of replacing some animal experiments in pain research with ethically conducted studies of human patients and healthy volunteers, in combination with in vitro methods. This report considers how a range of neuroimaging techniques including functional magnetic resonance imaging, magnetoencephalography and positron emission tomography, singly and combined, can address human pain conditions. In addition, microdialysis in human subjects; genome-wide association research, twin studies and other epidemiological approaches; and in vitro cell and tissue research, are examined for their replacement potential in combination with neuroimaging. Recommendations highlight further opportunities to advance the replacement of animal studies with robust methods of relevance to understanding and treating human pain.