Molecular ultrasound (US) imaging of overexpressed endothelial receptors has been shown to increase tumor visualization within a region-of-interest and is of particular interest in cancer research. This study used an in vivo animal model system for evaluating targeted microbubble (MB) contrast agents. 2LMP breast cancer-bearing mice (N = 6) expressed varying intratumoral levels of the target hemagglutinin (HA) receptor through adenoviral (Ad) vector modulation. Animals were administered a 'low' and then a 'high' Ad vector dose following initial US imaging. Anti-HA antibody-labeled or control MBs were prepared and molecular US imaging was performed 24 hours after Ad vector infection using a SONIX Tablet ultrasound system (Ultrasonix Medical Corp). Custom Matlab programs were developed to evaluate US image intensity from data collected using a MB destruction-replenishment technique. Results showed intratumoral enhancement due to targeted MB accumulation was significantly increased in the 'high' Ad vector dosed animals compared to the same animals administered a 'low' Ad vector dose (P = 0.001). Control MBs showed no significant differences between imaging days (P = 0.96). Additionally, targeted MBs on average produced a 10.5-fold increase in intratumoral image intensity for the 'low' HA receptor group and an 18.8-fold increase in image intensity for the 'high' HA receptor group compared to control MBs.