Currently, there are no applied molecular markers to aid in predicting risk of carcinoma in situ (CIS) progression to invasive cancer, and therefore, all women diagnosed with CIS undergo surgery. Standard assessment of protein expression in fixed tissue by immunohistochemistry (IHC) is not quantitative and hence is not well suited for measuring biomarkers. In this study, we developed an original analytical method for IHC quantification. Using our novel image-based uniplex (IBU) method, quantitative protein profiling was performed on 90 samples of the breast (17 histologically normal tissues, 16 benign lesions, 15 CIS, and 42 invasive carcinomas). Differences between groups were assessed using analysis of variance (ANOVA) and mixed effects models. Measuring protein expression on a continuous scale revealed a significant increase in Ras-related protein 1 (Rap1) and the insulin-like growth factor type I receptor (IGF-IR) in conjunction with the presence of cancer invasion. Women with invasive cancers were four times more likely to have increased levels of Rap1 [odds ratio (OR) = 3.91; P = 0.0002] and IGF-IR (OR=4.33; P<0.0001) than women with non-invasive lesions. Furthermore, expression of both proteins was also increased significantly in CIS adjacent to invasive tumors compared with non-cancerous tissue. These novel findings of a significant up-regulation of Rap1 and IGF-IR in CIS progressing to invasive cancers warrant further investigation of Rap1 and IGF-IR together as a dual biomarker to aid in predicting risk of progression and ultimately providing non-surgical treatment options to those at lower risk.