Although many details of T cell signaling have been described previously, a systematic, quantitative wide-scale analysis of global tyrosine phosphorylation events that are triggered by activation of the T cell receptor is lacking. Here we introduce and evaluate a new quantitative approach for phosphoproteomic analysis of signaling pathway structure. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or receptor stimulation time course experiments, often with restricted number of time points. Our approach combines genetic analysis of isogenic signaling pathway mutants with quantitative phosphoproteomic method that examines disruption of downstream phosphorylation events through a time course of receptor activation using recently developed visual pathway analysis tools. This new approach is evaluated in the context of the T cell signaling pathway and a T cell clone lacking the upstream Zap-70 tyrosine kinase and its reconstituted counterpart. In our approach, label free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Metabolic labeling of proteins using SILAC method allows for the calculation of ratios used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. Quantitative phosphoproteomic signatures indicative of downstream inhibition, downstream activation, and mutant compensation are revealed and validated with proteomic pathway visual analysis tools using established T cell signaling pathway structure as a scaffold.