Protein post-translational modifications (PTMs) tend to be dynamic and often occur at low stoichiometry, and thus can be challenging to identify and quantify. Despite these inherent challenges, PTM analyses (e.g., functional proteomics) can provide unprecedented insight into biological regulation in the context of health and disease, including identification of therapeutic drug targets, mechanisms of therapeutic resistance, and response to environmental perturbagens. Many cancer subtypes have poor response rates, poor overall survival statistics, and limited therapeutic options beyond chemotherapy. To identify potential therapeutic targets and therapeutic strategies for these tumors, we have applied functional proteomics with multiplexed isobaric tags (TMT) for quantification to gain insight into activated signaling networks. To enable reproducible quantification of low-abundance phosphorylation sites, in collaboration with ThermoFisher Scientific we have developed SureQuant pTyr 2.0, a method enabling the targeted quantification of ~1100 pTyr sites across almost all facets of cell biology. Application of TMT-based multiplexed phosphoproteomics and SureQuant pTyr 2.0 to cell lines and tumor specimens, including single sections or punches of frozen or FFPE-preserved tumor tissues, quantifies activated signaling networks with high sensitivity and reproducibility, enabling the identification of putative therapeutic targets from small amounts of starting material.