Dendritic cells (DCs) are increasingly prepared in vitro for use in clinical trials of human disease. Their utility in experimental immunotherapy has driven significant advances in the manufacture of these cells. Thus it has become imperative that, in concert with other quality control measures, a potency test be utilized for the GMP/GLP lot-release of DC products for preclinical and clinical studies. For this purpose we developed a novel method named the 'COSTIM bioassay', which selectively measures co-stimulatory activity, or functional potency of the DCs. In this method, T-cells stimulated with a sub-optimal amount of anti-CD3 antibody are unable to proliferate unless a source of co-stimulation (DCs) is added to the culture. We describe our validation of this method in this paper.