Extracellular signals are transmitted to intracellular targets thanks to a complex network of interacting proteins that regulate a large number of cellular processes. The signaling mechanisms that occur in CTLs are initiated by T cell receptors (TCRs) that recognize antigens presented in the groove of class I histocompatibility (MHC) molecules of target cells. The strength of this interaction and the consequent activation of intracellular mechanisms determine the fate of the developing T cells. The intracellular mediators of activation can be easily detected using a classical methodology such as western blot. Here we describe this technique bearing particular attention to these mediators using SNAP i.d., a newly developed protein detection system.