Knowledge of estrogen receptor (ER) and progesterone receptor (PR) status has been critical in the evolution of modern targeted therapy of breast cancer and remains essential for making informed therapeutic decisions. Recently, growth factor receptor HER2/neu (ERBB2) status has made it possible to provide another form of targeted therapy linked to the overexpression of this protein. Presently, pathologists determine the receptor status in formalin-fixed, paraffin-embedded sections using subjective, semiquantitative immunohistochemistry (IHC) assays and quantitative fluorescence in situ hybridization for HER2. We developed a single-tube multiplex TaqMan (mERPR+HER2) assay to quantitate mRNA levels of ER, PR, HER2, and two housekeeping genes for breast cancer formalin-fixed, paraffin-embedded sections. Using data from the discovery sample sets, we evaluated IHC-status-dependent cutoff-point and IHC-status-independent clustering methods for the classification of receptor status and then validated these results with independent sample sets. Compared with IHC-status, the accuracies of the mERPR+HER2 assay with the cutoff-point classification method were 0.98 (95% CI: 0.97-1.00), 0.92 (95% CI: 0.88-0.95), and 0.97 (95% CI: 0.95-0.99) for ER, PR, and HER2, respectively, for the validation sets. Furthermore, the areas under the receiver operating-characteristic curves were 0.997 (95% CI: 0.994-1.000), 0.967 (95% CI: 0.949-0.985), and 0.968 (95% CI: 0.915-1.000) for ER, PR, and HER2, respectively. This multiplex assay provides a sensitive and reliable method to quantitate hormonal and growth factor receptors.