T cell alloreactivity is responsible for much of the morbidity and mortality associated with tissue transplantation and graft versus host disease. Immunoassays for ex vivo monitoring and quantitation of alloreactive T cells are being increasingly utilised to provide valuable information for individualised clinical management of transplant recipients. Here we describe detailed methodologies for both traditional and novel assays utilised for the detection, quantitation, and functional characterisation of alloreactive T cells and highlight the key advantages and disadvantages of each system.