We investigated the potential of the pH-sensitive dye, CypHer5E, conjugated to Herceptin (pH-Her) for the sensitive detection of breast tumors in mice using noninvasive time-domain near-infrared fluorescence imaging and different methods of data analysis. First, the fluorescence properties of pH-Her were analyzed as function of pH and/or dye-to-protein ratio, and binding specificity was confirmed in cell-based assays. Subsequently, the performance of pH-Her in nude mice bearing orthotopic HER2-positive (KPL-4) and HER2-negative (MDA-MB-231) breast carcinoma xenografts was compared to that of an always-on fluorescent conjugate Alexa Fluor 647-Herceptin (Alexa-Her). Subtraction of autofluorescence and lifetime (LT)-gated image analyses were performed for background fluorescence suppression. In mice bearing HER2-positive tumors, autofluorescence subtraction together with the selective fluorescence enhancement of pH-Her solely in the tumor's acidic environment provided high contrast-to-noise ratios (CNRs). This led to an improved sensitivity of tumor detection compared to Alexa-Her. In contrast, LT-gated imaging using LTs determined in model systems did not improve tumor-detection sensitivity in vivo for either probe. In conclusion, pH-Her is suitable for sensitive in vivo monitoring of HER2-expressing breast tumors with imaging in the intensity domain and represents a promising tool for detection of weak fluorescent signals deriving from small tumors or metastases.