A systematic comparison of the immunostimulatory capacity of TLR 2, 3, 4, 5, 7 and 9 agonists and an agonistic CD40-specific antibody was performed in a single long peptide vaccination model. All adjuvants activated DC in vitro but not all induced a strong functional T-cell response in vivo. Optimal clonal CD8+ T-cell expansion depended on the capacity of agonists to mature pro-inflammatory DC and the duration of their in vivo stimulatory effect. Strong agonists promoted the induction of both antigen-specific IFNγ-producing CD4+ T-helper cells and high numbers of IFNγ producing CD8+ effector T-cells that killed target cells in vivo. Importantly, the capacity of an agonist to function as an adjuvant depended on the vaccine strategy used. Collectively, the multi-parameter system presented here can be used as a general road map to develop therapeutic vaccines.