Multiprotein complexes play an essential role in the propagation and integration of cellular signals. However, systems level analyses of signaling-dependent changes in the pattern of molecular interactions are still missing. Signaling in T lymphocytes is one prominent example, in which multiprotein complexes orchestrate signal transduction. We have implemented peptide microarrays comprising a set of interaction motifs of signaling proteins for network-based analyses of signaling-dependent changes in molecular interactions. Lysates of resting or stimulated cells are incubated on these arrays and the binding of signaling proteins is detected by immunofluorescence. Signaling-dependent complex formation leads to changes of signals on the microarrays in two ways: (1) Masking of a binding site of a signaling protein for a peptide on the array results in a signal decrease. (2) Interaction of a protein with a second protein, that in turn binds to a peptide on the array results in a signal increase for the first protein. Dissipation of complexes leads to the reverse changes. Competition with peptides corresponding to interaction motifs provides detailed information on the architecture of complexes; lack of individual signaling proteins reveals the functional interdependence of interactions in the network. We show that complex formation through phosphorylation of the scaffolding protein LAT acts as a signal amplifier. PLC1 deficiency increases the resting state levels of LAT-dependent complexes and augments the recruitment of the phosphatase SHPTP2 into complexes. For the analysis of signaling networks, the parallel detection of changes in interactions enables the identification of functional interdependencies with minimum a priori knowledge.