Notch receptors regulate cell fate determination, stem cell self-renewal, proliferation and apoptosis. We previously reported that elevated mRNA expression of the Notch ligand JAG1 identifies breast cancer patients with a poor prognosis. Here we show through immunohistochemical analysis of the same breast cancer cases (N=127) that patients with tumors expressing high levels of JAG1 protein had a worse outcome than those with tumors expressing low levels (10-year survival 26 vs 48%, and median survival 63 vs 108 months, respectively; P=0.03). We also describe the novel application of the Allred score to quantify JAG1 mRNA and protein expression levels. Using the Allred score, patients with tumors expressing high levels of JAG1 mRNA had a worse outcome than those with tumors expressing low levels (10-year survival 16 vs 47%, and median survival 43 months vs 100 months, respectively; P<0.001). Interestingly, when tumors were classified as either high or low for JAG1 mRNA or protein expression, there was only 65% agreement (kappa=0.08) between the two methods of expression analysis. When JAG1 mRNA and protein data were combined, patients with tumors expressing low levels of both had a 10-year survival of 53% and median survival of 131 months. In comparison, patients with tumors expressing either high levels of JAG1 protein, mRNA or both had reduced 10-year survival and median survival (31%, 19%, 11% and 77, 43, 23 months respectively; P<0.0001). There was marginal evidence of an interaction effect (P=0.055), which indicated that the prognostic value of JAG1 protein was limited to the JAG1 mRNA-low subgroup. These data show that the Allred score can be used to rapidly quantify JAG1 mRNA and protein levels in breast cancer to identify patients who have a significant survival disadvantage and who may benefit from therapies (such as gamma-secretase inhibitors) that target signaling through the Notch pathway.