Targeted therapies have often given disappointing results when used as single agents in solid tumors, suggesting the importance of devising rational combinations of targeted drugs. We hypothesized that construction of such combinations could be guided by identification of growth and survival pathways whose activity or expression become upregulated in response to single-agent drug treatment. We mapped alterations in signaling pathways assessed by gene array and protein phosphorylation to identify compensatory signal transduction pathways in prostate cancer xenografts treated with a MAP/ERK kinase (MEK) inhibitor PD325901. In addition to numerous components of the extracellular signal–regulated kinase (ERK) signaling pathway, components of the IKK, hedgehog, and phosphoinositide 3-kinase/Akt/mTOR pathways were upregulated following treatment with PD325901. Combinations of PD325901 with inhibitors of any one of these upregulated pathways provided synergistically greater growth inhibition of in vitro cell growth and survival than the individual drugs alone. Thus, the identification of compensatory signal transduction pathways paves the way for rational combinatorial therapies for the effective treatment of prostate cancer. Mol Cancer Ther; 10(9); 1581–90. ©2011 AACR.