Identifying factors responsible for variation in drug response is essential for the effective use of targeted therapeutics. We profiled signaling pathway activity in a collection of breast cancer cell lines before and after stimulation with physiologically relevant ligands, which revealed the variability in network activity among cells of known genotype and molecular subtype. Despite the receptor-based classification of breast cancer subtypes, we found that the abundance and activity of signaling proteins in unstimulated cells (basal profile), as well as the activity of proteins in stimulated cells (signaling profile), varied within each subtype. Using a partial least squares regression approach, we constructed models that significantly predicted sensitivity to 23 targeted therapeutics. This analysis identified key proteins that could serve as biomarkers of drug sensitivity. For example, one model showed that the response to the growth factor receptor ligand heregulin effectively predicted the sensitivity of cells to drugs targeting the cell survival pathway mediated by PI3K (phosphoinositide 3-kinase) and Akt; whereas the abundance of Akt or the mutational status of the enzymes in the pathway did not. Thus, basal and signaling protein profiles may yield new biomarkers and enable the identification of appropriate therapies in cancers characterized by similar functional dysregulation of signaling networks.