Intratumoral heterogeneity of signaling networks may contribute to targeted cancer therapy resistance, including in the highly lethal brain cancer glioblastoma (GBM). We performed single-cell phosphoproteomics on a patient-derived in vivo GBM model of mTOR kinase inhibitor resistance and coupled it to an analytical approach for detecting changes in signaling coordination. Alterations in the protein signaling coordination were resolved as early as 2.5 days after treatment, anticipating drug resistance long before it was clinically manifest. Combination therapies were identified that resulted in complete and sustained tumor suppression in vivo. This approach may identify actionable alterations in signal coordination that underlie adaptive resistance, which can be suppressed through combination drug therapy, including non-obvious drug combinations. Display omitted •Sequencing excludes the selection of an mTORki-resistant genotype in a GBM model•Heterogeneous signaling networks rapidly adapt to mTORki to drive resistance•Resistance-promoting signaling is observed a priori by single-cell analysis•Network analyses point to therapy combinations for long-term disease suppression Wei et al. utilize single-cell phosphoproteomic analysis of patient-derived glioblastoma models to identify shifts in signaling coordination following short-term treatment with kinase inhibitors, which facilitates the design of combination therapy approaches with reduced resistance and improved efficacy.