Subsets of T cells can be distinguished on basis of their cytokine production and secretion profile. With the critical role of cytokines in the regulation of immune and inflammatory responses, cytokines hold the promise to become the ideal biomarkers to monitor development and progression of immune-mediated diseases, study the development of new therapeutic approaches (both in vitro and in vivo) and as outcome parameters. Because of the numerous interactions in the cytokine network, the pleiotropic actions and redundancy, it will be necessary to monitor the complete spectrum of cytokines. As such, the multiplex immunoassay (MIA) is the ideal technique for that purpose. This paper reviews the critical methodological steps of this technique, including the procedures for antibody coupling to beads and matrix effects of biological fluids and buffer solutions. The power and robustness of the MIA technique is illustrated by an analysis of cytokine profiles in juvenile idiopathic arthritis.